Optimized Fc variants and methods for their generation

ABSTRACT

The present invention relates to optimized Fc variants, methods for their generation, and antibodies and Fc fusions comprising optimized Fc variants.

This application claims benefit under 35 U.S.C. §199(e) to U.S. Pat.Nos. 10/672280, filed Sep. 26, 2003, 10/379392, filed Mar. 3, 2003,60/477,839 filed Jun. 12, 2003; 60/467,606, filed May 2, 2003;60/414,433 filed Sep. 27, 2002; and 60/442,301 filed Jan. 23, 2003, allof which are expressly incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel optimized Fc variants,engineering methods for their generation, and their application,particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgA1 and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing features between these antibody classes aretheir constant regions, although subtler differences may exist in the Vregion. FIG. 1 shows an IgG1 antibody, used here as an example todescribe the general structural features of immunoglobulins. IgGantibodies are tetrameric proteins composed of two heavy chains and twolight chains. The IgG heavy chain is composed of four immunoglobulindomains linked from N- to C-terminus in the order VH-Cγ1-Cγ2-Cγ3,referring to the heavy chain variable domain, constant gamma 1 domain,constant gamma 2 domain, and constant gamma 3 domain respectively. TheIgG light chain is composed of two immunoglobulin domains linked from N-to C-terminus in the order V_(L)-C_(L), referring to the light chainvariable domain and the light chain constant domain respectively.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. The majority of sequence variability occurs in thecomplementarity determining regions (CDRs). There are 6 CDRs total,three each per heavy and light chain, designated V_(H) CDR1, V_(H) CDR2,V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, and V_(L) CDR3. The variable regionoutside of the CDRs is referred to as the 6 CDRs total, three each perheavy and light chain, designated V_(H) CDR1, V_(H) CDR2, V_(H) CDR3,V_(L) FR region between different antibodies. Overall, thischaracteristic architecture of antibodies provides a stable scaffold(the FR region) upon which substantial antigen binding diversity (theCDRs) can be explored by the immune system to obtain specificity for abroad array of antigens. A number of high-resolution structures areavailable for a variety of variable region fragments from differentorganisms, some unbound and some in complex with antigen. The sequenceand structural features of antibody variable regions are wellcharacterized (Morea et al, 1997, Biophys Chem 68:9-16; Morea et al.,2000, Methods 20:267-279), and the conserved features of antibodies haveenabled the development of a wealth of antibody engineering techniques(Maynard et at., 2000, Annu Rev Biomed Eng 2:339-376). For example, itis possible to graft the CDRs from one antibody, for example a murineantibody, onto the framework region of another antibody, for example ahuman antibody. This process, referred to in the art as “humanization”,enables generation of less immunogenic antibody therapeutics fromnonhuman antibodies. Fragments comprising the variable region can existin the absence of other regions of the antibody, including for examplethe antigen binding fragment (Fab) comprising V_(H)-Cγ1 and V_(H)-C_(L),the variable fragment (Fv) comprising V_(H) and V_(L), the single chainvariable fragment (scFv) comprising V_(H) and V_(L) linked together inthe same chain, as well as a variety of other variable region fragments(Little et al., 2000, Immunol Today 21:364-370).

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region, as shown inFIG. 1, comprises Ig domains Cγ2 and Cγ3 and the N-terminal hingeleading into Cγ2. An important family of Fc receptors for the IgG classare the Fc gamma receptors (FcγRs). These receptors mediatecommunication between antibodies and the cellular arm of the immunesystem (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220;Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans thisprotein family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). Thesereceptors typically have an extracellular domain that mediates bindingto Fc, a membrane spanning region, and an intracellular domain that maymediate some signaling event within the cell. These receptors areexpressed in a variety of immune cells including monocytes, macrophages,neutrophils, dendritic cells, eosinophils, mast cells, platelets, Bcells, large granular lymphocytes, Langerhans' cells, natural killer(NK) cells, and γγ T cells. Formation of the Fc/FcγR complex recruitsthese effector cells to sites of bound antigen, typically resulting insignaling events within the cells and important subsequent immuneresponses such as release of inflammation mediators, B cell activation,endocytosis, phagocytosis, and cytotoxic attack. The ability to mediatecytotoxic and phagocytic effector functions is a potential mechanism bywhich antibodies destroy targeted cells. The cell-mediated reactionwherein nonspecific cytotoxic cells that express FcγRs recognize boundantibody on a target cell and subsequently cause lysis of the targetcell is referred to as antibody dependent cell-mediated cytotoxicity(ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetieet al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, AnnuRev Immunol 19:275-290). The cell-mediated reaction wherein nonspecificcytotoxic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause phagocytosis of the target cell is referredto as antibody dependent cell-mediated phagocytosis (ADCP). A number ofstructures have been solved of the extracellular domains of human FcγRs,including FcγRIIa (pdb accession code 1H9V)(Sondermann et al., 2001, JMol Biol 309:737-749) (pdb accession code 1FCG)(Maxwell et al., 1999,Nat Struct Biol 6:437442), FcγRIIb (pdb accession code 2FCB)(Sondermannet al., 1999, Embo J 18:1095-1103); and FcγRIIIb (pdb accession code1E4J)(Sondermann et al., 2000, Nature 406:267-273.). All FcγRs bind thesame region on Fc, at the N-terminal end of the Cγ2 domain and thepreceding hinge, shown in FIG. 2. This interaction is well characterizedstructurally (Sondermann et al., 2001, J Mol Biol 309:737-749), andseveral structures of the human Fc bound to the extracellular domain ofhuman FcγRIIIb have been solved (pdb accession code 1E4K)(Sondermann etal., 2000, Nature 406:267-273.) (pdb accession codes 1IIS and1IIX)(Radaev et al., 2001, J Biol Chem 276:16469-16477), as well as hasthe structure of the human IgE Fc/FcεRIα complex (pdb accession code1F6A)(Garman et al., 2000, Nature 406:259-266).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65). All FcγRs bind the same region on IgG Fc, yet with differentaffinities: the high affinity binder FcγRI has a Kd for IgG1 of 10⁻⁸M⁻¹, whereas the low affinity receptors FcγRII and FcγRIII generallybind at 10⁻⁶ and 10⁻⁵ respectively. The extracellular domains ofFcγRIIIa and FcγRIIIb are 96% identical, however FcγRIIIb does not havea intracellular signaling domain. Furthermore, whereas FcγRI, FcγRIIa/c,and FcγRIIIa are positive regulators of immune complex-triggeredactivation, characterized by having an intracellular domain that has animmunoreceptor tyrosine-based activation motif (ITAM), FcγRIIb has animmunoreceptor tyrosine-based inhibition motif (ITIM) and is thereforeinhibitory. Thus the former are referred to as activation receptors, andFcγRIIb is referred to as an inhibitory receptor. The receptors alsodiffer in expression pattern and levels on different immune cells. Yetanother level of complexity is the existence of a number of FcγRpolymorphisms in the human proteome. A particularly relevantpolymorphism with clinical significance is V158/F158 FcγRIIIa. HumanIgG1 binds with greater affinity to the V158 allotype than to the F158allotype. This difference in affinity, and presumably its effect on ADCCand/or ADCP, has been shown to be a significant determinant of theefficacy of the anti-CD20 antibody rituximab (Rituxan®, a registeredtrademark of IDEC Pharmaceuticals Corporation). Patients with the V158allotype respond favorably to rituximab treatment; however, patientswith the lower affinity F158 allotype respond poorly (Cartron et al.,2002, Blood 99:754-758). Approximately 10-20% of humans are V158/V158homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans areF158/F158 homozygous (Lehrnbecher et al., 1999, Blood 94:42204232;Cartron et al., 2002, Blood 99:754-758). Thus 80-90% of humans are poorresponders, that is they have at least one allele of the F158 FcγRIIIa.

An overlapping but separate site on Fc, shown in FIG. 1, serves as theinterface for the complement protein C1q. In the same way that Fc/FcγRbinding mediates ADCC, Fc/C1q binding mediates complement dependentcytotoxicity (CDC). C1q forms a complex with the serine proteases C1rand C1s to form the C1 complex. C1q is capable of binding sixantibodies, although binding to two IgGs is sufficient to activate thecomplement cascade. Similar to Fc interaction with FcγRs, different IgGsubclasses have different affinity for C1q, with IgG1 and IgG3 typicallybinding substantially better to the FcγRs than IgG2 and IgG4 (Jefferiset al., 2002, Immunol Lett 82:57-65). There is currently no structureavailable for the Fc/C1q complex; however, mutagenesis studies havemapped the binding site on human IgG for C1q to a region involvingresidues D270, K322, K326, P329, and P331, and E333 (Idusogie et al.,2000, J Immunol 164:4178-4184; Idusogie et al., 2001, J Immunol166:2571-2575).

A site on Fc between the Cγ2 and Cγ3 domains, shown in FIG. 1, mediatesinteraction with the neonatal receptor FcRn, the binding of whichrecycles endocytosed antibody from the endosome back to the bloodstream(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie etal., 2000, Annu Rev Immunol 18:739-766). This process, coupled withpreclusion of kidney filtration due to the large size of the full lengthmolecule, results in favorable antibody serum half-lives ranging fromone to three weeks. Binding of Fc to FcRn also plays a key role inantibody transport. The binding site for FcRn on Fc is also the site atwhich the bacterial proteins A and G bind. The tight binding by theseproteins is typically exploited as a means to purify antibodies byemploying protein A or protein G affinity chromatography during proteinpurification. Thus the fidelity of this region on Fc is important forboth the clinical properties of antibodies and their purification.Available structures of the rat Fc/FcRn complex (Martin et al., 2001,Mol Cell 7:867-877), and of the complexes of Fc with proteins A and G(Deisenhofer, 1981, Biochemistry 20:2361-2370; Sauer-Eriksson et al.,1995, Structure 3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol5:471-481) provide insight into the interaction of Fc with theseproteins.

A key feature of the Fc region is the conserved N-linked glycosylationthat occurs at N297, shown in FIG. 1. This carbohydrate, oroligosaccharide as it is sometimes referred, plays a critical structuraland functional role for the antibody, and is one of the principlereasons that antibodies must be produced using mammalian expressionsystems. While not wanting to be limited to one theory, it is believedthat the structural purpose of this carbohydrate may be to stabilize orsolubilize Fc, determine a specific angle or level of flexibilitybetween the Cγ3 and Cγ2 domains, keep the two Cγ2 domains fromaggregating with one another across the central axis, or a combinationof these. Efficient Fc binding to FcγR and C1q requires thismodification, and alterations in the composition of the N297carbohydrate or its elimination affect binding to these proteins (Umañaet al., 1999, Nat Biotechnol 17:176-180; Davies et a., 2001, BiotechnolBioeng 74:288-294; Mimura et a., 2001, J Biol Chem 276:45539-45547.;Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001,J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem277:26733-26740; Simmons et al., 2002, J Immunol Methods 263:133-147).Yet the carbohydrate makes little if any specific contact with FcγRs(Radaev et al., 2001, J Biol Chem 276:16469-16477), indicating that thefunctional role of the N297 carbohydrate in mediating Fc/FcγR bindingmay be via the structural role it plays in determining the Fcconformation. This is supported by a collection of crystal structures offour different Fc glycoforms, which show that the composition of theoligosaccharide impacts the conformation of Cγ2 and as a result theFc/FcγR interface (Krapp et al., 2003, J Mol Biol 325:979-989).

The features of antibodies discussed above—specificity for target,ability to mediate immune effector mechanisms, and long half-life inserum—make antibodies powerful therapeutics. Monoclonal antibodies areused therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, and cardiovascular disease. There arecurrently over ten antibody products on the market and hundreds indevelopment. In addition to antibodies, an antibody-like protein that isfinding an expanding role in research and therapy is the Fc fusion(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200). An Fc fusion is a protein whereinone or more polypeptides is operably linked to Fc. An Fc fusion combinesthe Fc region of an antibody, and thus its favorable effector functionsand pharmacokinetics, with the target-binding region of a receptor,ligand, or some other protein or protein domain. The role of the latteris to mediate target recognition, and thus it is functionally analogousto the antibody variable region. Because of the structural andfunctional overlap of Fc fusions with antibodies, the discussion onantibodies in the present invention extends directly to Fc fusions.

Despite such widespread use, antibodies are not optimized for clinicaluse. Two significant deficiencies of antibodies are their suboptimalanticancer potency and their demanding production requirements. Thesedeficiencies are addressed by the present invention

There are a number of possible mechanisms by which antibodies destroytumor cells, including anti-proliferation via blockage of needed growthpathways, intracellular signaling leading to apoptosis, enhanced downregulation and/or turnover of receptors, CDC, ADCC, ADCP, and promotionof an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol11:541-547; Glennie et al., 2000, Immunol Today 21:403-410). Anti-tumorefficacy may be due to a combination of these mechanisms, and theirrelative importance in clinical therapy appears to be cancer dependent.Despite this arsenal of anti-tumor weapons, the potency of antibodies asanti-cancer agents is unsatisfactory, particularly given their highcost. Patient tumor response data show that monoclonal antibodiesprovide only a small improvement in therapeutic success over normalsingle-agent cytotoxic chemotherapeutics. For example, just half of allrelapsed low-grade non-Hodgkin's lymphoma patients respond to theanti-CD20 antibody rituximab (McLaughlin et al., 1998, J Clin Oncol16:2825-2833). Of 166 clinical patients, 6% showed a complete responseand 42% showed a partial response, with median response duration ofapproximately 12 months. Trastuzumab (Herceptin®, a registered trademarkof Genentech), an anti-HER2/neu antibody for treatment of metastaticbreast cancer, has less efficacy. The overall response rate usingtrastuzumab for the 222 patients tested was only 15%, with 8 completeand 26 partial responses and a median response duration and survival of9 to 13 months (Cobleigh et al., 1999, J Clin Oncol 17:2639-2648).Currently for anticancer therapy, any small improvement in mortalityrate defines success. Thus there is a significant need to enhance thecapacity of antibodies to destroy targeted cancer cells.

A promising means for enhancing the anti-tumor potency of antibodies isvia enhancement of their ability to mediate cytotoxic effector functionssuch as ADCC, ADCP, and CDC. The importance of FcγR-mediated effectorfunctions for the anti-cancer activity of antibodies has beendemonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sci USA95:652-656; Clynes et al., 2000, Nat Med 6:443-446), and the affinity ofinteraction between Fc and certain FcγRs correlates with targetedcytotoxicity in cell-based assays (Shields et al., 2001, J Biol Chem276:6591-6604; Presta et al., 2002, Biochem Soc Trans 30:487-490;Shields et al., 2002, J Biol Chem 277:26733-26740). Additionally, acorrelation has been observed between clinical efficacy in humans andtheir allotype of high (V158) or low (F158) affinity polymorphic formsof FcγRIIIa (Cartron et a!., 2002, Blood 99:754-758). Together thesedata suggest that an antibody with an Fc region optimized for binding tocertain FcγRs may better mediate effector functions and thereby destroycancer cells more effectively in patients. The balance betweenactivating and inhibiting receptors is an important consideration, andoptimal effector function may result from an Fc with enhanced affinityfor activation receptors, for example FcγRI, FcγRIIa/c, and FcγRIIIa,yet reduced affinity for the inhibitory receptor FcγRIIb. Furthermore,because FcγRs can mediate antigen uptake and processing by antigenpresenting cells, enhanced Fc/FcγR affinity may also improve thecapacity of antibody therapeutics to elicit an adaptive immune response.

Mutagenesis studies have been carried out on Fc towards various goals,with substitutions typically made to alanine (referred to as alaninescanning) or guided by sequence homology substitutions (Duncan et al.,1988, Nature 332:563-564; Lund et al., 1991, J Immunol 147:2657-2662;Lund et al., 1992, Mol Immunol 29:53-59; Jefferis et al., 1995, ImmunolLett 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al.,1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Shieldset al., 2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, ImmunolLett 82:57-65) (U.S. Pat. Nos. 5,624,821; 5,885,573; PCT WO 00/42072;PCT WO 99/58572). The majority of substitutions reduce or ablate bindingwith FcγRs. However some success has been achieved at obtaining Fcvariants with higher FcγR affinity. (See for example U.S. Pat. No.5,624,821, and PCT WO 00/42072). For example, Winter and colleaguessubstituted the human amino acid at position 235 of mouse IgG2b antibody(a glutamic acid to leucine mutation) that increased binding of themouse antibody to human FcγRI by100-fold (Duncan et al., 1988, Nature332:563-564) (U.S. Pat. No. 5,624,821). Shields et al. used alaninescanning mutagenesis to map Fc residues important to FcγR binding,followed by substitution of select residues with non-alanine mutations(Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al., 2002,Biochem Soc Trans 30:487-490) (PCT WO 00/42072). Several mutationsdisclosed in this study, including S298A, E333A, and K334A, showenhanced binding to the activating receptor FcγRIIIa and reduced bindingto the inhibitory receptor FcγRIIb. These mutations were combined toobtain double and triple mutation variants that show additiveimprovements in binding. The best variant disclosed in this study is aS298A/E333ANK334A triple mutant with approximately a 1.7-fold increasein binding to F158 FcγIIIa, a 5-fold decrease in binding to FcγRIIb, anda 2.1-fold enhancement in ADCC.

Enhanced affinity of Fc for FcγR has also been achieved using engineeredglycoforms generated by expression of antibodies in engineered orvariant cell lines (Umaña et al., 1999, Nat Biotechnol 17:176-180;Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002,J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473). This approach has generated substantial enhancements ofthe capacity of antibodies to bind FcγRIIIa and to mediate ADCC.Although there are practical limitations such as the growth efficiencyof the expression strains under large scale production conditions, thisapproach for enhancing Fc/FcγR affinity and effector function ispromising. Indeed, coupling of these alternate glycoform technologieswith the Fc variants of the present invention may provide additive orsynergistic effects for optimal effector function.

Although there is a need for greater effector function, for someantibody therapeutics reduced or eliminated effector function may bedesired. This is often the case for therapeutic antibodies whosemechanism of action involves blocking or antagonism but not killing ofthe cells bearing target antigen. In these cases depletion of targetcells is undesirable and can be considered a side effect. For example,the ability of anti-CD4 antibodies to block CD4 receptors on T cellsmakes them effective anti-inflammatories, yet their ability to recruitFcγR receptors also directs immune attack against the target cells,resulting in T cell depletion (Reddy et al., 2000, J Immunol164:1925-1933). Effector function can also be a problem for radiolabeledantibodies, referred to as radioconjugates, and antibodies conjugated totoxins, referred to as immunotoxins. These drugs can be used to destroycancer cells, but the recruitment of immune cells via Fc interactionwith FcγRs brings healthy immune cells in proximity to the deadlypayload (radiation or toxin), resulting in depletion of normal lymphoidtissue along with targeted cancer cells (Hutchins et al., 1995, ProcNatl Acad Sci USA 92:11980-11984; White et al., 2001, Annu Rev Med52:125-145). This problem can potentially be circumvented by using IgGisotypes that poorly recruit complement or effector cells, for exampleIgG2 and IgG4. An alternate solution is to develop Fc variants thatreduce or ablate binding (Alegre et al., 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA92:11980-11984; Armour et al., 1999, Eur J Immunol 29:2613-2624; Reddyet al., 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol200:16-26; Shields et al., 2001, J Biol Chem 276:6591-6604) (U.S. Pat.Nos. 6,194,551; 5,885,573; PCT WO 99/58572). A critical considerationfor the reduction or elimination of effector function is that otherimportant antibody properties not be perturbed. Fc variants should beengineered that not only ablate binding to FcγRs and/or C1q, but alsomaintain antibody stability, solubility, and structural integrity, aswell as ability to interact with other important Fc ligands such as FcRnand proteins A and G.

The present invention addresses another major shortcoming of antibodies,namely their demanding production requirements (Garber, 2001, NatBiotechnol 19:184-185; Dove, 2002, Nat Biotechnol 20:777-779).Antibodies must be expressed in mammalian cells, and the currentlymarketed antibodies together with other high-demand biotherapeuticsconsume essentially all of the available manufacturing capacity. Withhundreds of biologics in development, the majority of which areantibodies, there is an urgent need for more efficient and cheapermethods of production. The downstream effects of insufficient antibodymanufacturing capacity are three-fold. First, it dramatically raises thecost of goods to the producer, a cost that is passed on to the patient.Second, it hinders industrial production of approved antibody products,limiting availability of high demand therapeutics to patients. Finally,because clinical trials require large amounts of a protein that is notyet profitable, the insufficient supply impedes progress of the growingantibody pipeline to market.

Alternative production methods have been explored in attempts atalleviating this problem. Transgenic plants and animals are beingpursued as potentially cheaper and higher capacity production systems(Chadd et al., 2001, Curr Opin Biotechnol 12:188-194). Such expressionsystems, however, can generate glycosylation patterns significantlydifferent from human glycoproteins. This may result in reduced or evenlack of effector function because, as discussed above, the carbohydratestructure can significantly impact FcγR and complement binding. Apotentially greater problem with nonhuman glycoforms may beimmunogenicity; carbohydrates are a key source of antigenicity for theimmune system, and the presence of nonhuman glycoforms has a significantchance of eliciting antibodies that neutralize the therapeutic, or worsecause adverse immune reactions. Thus the efficacy and safety ofantibodies produced by transgenic plants and animals remains uncertain.Bacterial expression is another attractive solution to the antibodyproduction problem. Expression in bacteria, for example E. coli,provides a cost-effective and high capacity method for producingproteins. For complex proteins such as antibodies there are a number ofobstacles to bacterial expression, including folding and assembly ofthese complex molecules, proper disulfide formation, and solubility,stability, and functionality in the absence of glycosylation becauseproteins expressed in bacteria are not glycosylated. Full lengthunglycosylated antibodies that bind antigen have been successfullyexpressed in E. coli (Simmons et al., 2002, J Immunol Methods263:133-147), and thus, folding, assembly, and proper disulfideformation of bacterially expressed antibodies are possible in theabsence of the eukaryotic chaperone machinery. However the ultimateutility of bacterially expressed antibodies as therapeutics remainshindered by the lack of glycosylation, which results in lack effectorfunction and may result in poor stability and solubility. This willlikely be more problematic for formulation at the high concentrationsfor the prolonged periods demanded by clinical use.

An aglycosylated Fc with favorable solution properties and the capacityto mediate effector functions would be significantly enabling for thealternate production methods described above. By overcoming thestructural and functional shortcomings of aglycosylated Fc, antibodiescan be produced in bacteria and transgenic plants and animals withreduced risk of immunogenicity, and with effector function for clinicalapplications in which cytotoxicity is desired such as cancer. Thepresent invention describes the utilization of protein engineeringmethods to develop stable, soluble Fc variants with effector function.Currently, such Fc variants do not exist in the art.

In summary, there is a need for antibodies with enhanced therapeuticproperties. Engineering of optimized or enhanced Fc variants is apromising approach to meeting this need. Yet a substantial obstacle toengineering Fc variants with the desired properties is the difficulty inpredicting what amino acid modifications, out of the enormous number ofpossibilities, will achieve the desired goals, coupled with theinefficient production and screening methods for antibodies. Indeed oneof the principle reasons for the incomplete success of the prior art isthat approaches to Fc engineering have thus far involved hit-or-missmethods such as alanine scans or production of glycoforms usingdifferent expression strains. In these studies, the Fc modificationsthat were made were fully or partly random in hopes of obtainingvariants with favorable properties. The present invention provides avariety of engineering methods, many of which are based on moresophisticated and efficient techniques, which may be used to overcomethese obstacles in order to develop Fc variants that are optimized forthe desired properties. The described engineering methods provide designstrategies to guide Fc modification, computational screening methods todesign favorable Fc variants, library generation approaches fordetermining promising variants for experimental investigation, and anarray of experimental production and screening methods for determiningthe Fc variants with favorable properties.

SUMMARY OF THE INVENTION

The present invention provides Fc variants that are optimized for anumber of therapeutically relevant properties. These Fc variants aregenerally contained within a variant protein, that preferably comprisesan antibody or a Fc fusion protein.

It is an object of the present invention to provide novel Fc positionsat which amino acid modifications may be made to generate optimized Fcvariants. Said Fc positions include 230, 240, 244, 245, 247, 262, 263,266, 273, 275, 299, 302, 313, 323, 325, 328, and 332, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. The present invention describes any amino acid modification atany of said novel Fc positions in order to generate an optimized Fcvariant.

It is a further object of the present invention to provide Fc variantsthat have been screened computationally. A computationally screened Fcvariant is one that is predicted by the computational screeningcalculations described herein as having a significantly greaterpotential than random for being optimized for a desired property. Inthis way, computational screening serves as a prelude to or surrogatefor experimental screening, and thus said computationally screened Fcvariants are considered novel.

It is a further object of the present invention to provide Fc variantsthat have been characterized using one or more of the experimentalmethods described herein. In one embodiment, said Fc variants compriseat least one amino acid substitution at a position selected from thegroup consisting of: 230, 233, 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 267, 269, 270, 272, 273, 274, 275, 276,278, 283, 296, 297, 298, 299, 302, 313, 318, 320, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, and 335, wherein the numberingof the residues in the Fc region is that of the EU index as in Kabat. Inone embodiment, said Fc variants comprise at least one amino acidsubstitution at a position selected from the group consisting of: 221,222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239,240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281,283, 285, 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300,302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335 336 and 428, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. In apreferred embodiment, said Fc variants comprise at least onesubstitution selected from the group consisting of P230A, E233D, L234D,L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D,L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D,S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T,V240M, F241W, F241L, F241Y, F241E, F241R, F243W, F243L F243Y, F243R,F243Q, P244H, P245A, P247V, P247G, V2621, V262A, V262T, V262E, V263I,V263A, V263T, V263M, V264L, V264I, V264W, V264T, V264R, V264F, V264M,V264Y, V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L,D265H, D265T, V266I, V266A, V266T, V266M, S267Q, S267L, S267T, S267H,S267D, S267N, E269H, E269Y, E269F, E269R, E269T, E269L, E269N, D270Q,D270T, D270H, E272S, E272K, E272I, E272Y, V273I, K274T, K274E, K274R,K274L, K274Y, F275W, N276S, N276E, N276R, N276L, N276Y, Y278T, Y278E,Y278K, Y278W, E283R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T, Y296L,Y296I, Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A, T299S,T299V, T299H, T299F, T299E, V302I, W313F, E318R, K320T, K320D, K320I,K322T, K322H, V323I, S324T, S324D, S324R, S324I, S324V, S324L, S324Y,N325Q, N325L, N325I, N325D, N325E, N325A, N325T, N325V, N325H, K326L,K326I, K326T, A327N, A327L, A327D, A327T, L328M, L328D, L328E, L328N,L328Q, L328F, L328I, L328V, L328T, L328H, L328A, P329F, A330L, A330Y,A330V, A330I, A330F, A330R, A330H, A330S, A330W, A330M, P331V, P331H,I332D, I332E, I332N, I332Q, I332T, I332H, 1332Y, I332A, E333T, E333H,E333I, E333Y, K334I, K334T, K334F, T335D, T335R, and T335Y, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. In a mostly preferred embodiment, said Fc variants are selectedfrom the group consisting of V264L, V264I, F241W, F241L, F243W, F243L,F241L/F243L/V262I/V264I, F241W/F243W, F241W/F243W/V262A/V264A,F241L/V262I, F243L/V264I, F243L/V262I/V264W, F241Y/F243Y/V262T/V264T,F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E, L328F,I332E, L328M/I332E, P244H, P245A, P247V, W313F, P244H/P245A/P247V,P247G, V264I/I1332E, F241E/F243R/V262E/V264R/1332E,F241E/F243Q/V262T/V264E/1332E, F241R/F243Q/V262T/V264R/I332E,F241E/F243Y/V262T/V264R/I332E, S298A/1332E, S239E/I332E, S239Q/I332E,S239E, D265G, D265N, S239E/D265G, S239E/D265N, S239E/D265Q, Y296E,Y296Q, T299I, A327N, S267Q/A327S, S267L/A327S, A327L, P329F, A330L,A330Y, I332D, N297S, N297D, N297S/I332E, N297D/I332E, N297E/I332E,D265Y/N297D/I332E, D265Y/N297D/T299L/I332E, D265F/N297E/I332E,L328I/I332E, L328Q/I332E, I332N, I332Q, V264T, V264F, V240I, V263,V266I, T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N, S239F,S239D/I332D, S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D,S239E/I332N, S239E/I332Q, S239N/I332D, S239N/I332E, S239N/I332N,S239N/I332Q, S239Q/I332D, S239Q/I332N, S239Q/I332Q, Y296D, Y296N,F241Y/F243Y/V262T/V264T/N297D/I332E, A330Y/I332E, V264I/A330Y/I332E,A330L/I332E, V264I/A330L/I332E, L234D, L234D, L234E, L234Q, L234T,L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T,L235H, L235Y, L235I, L235V, L235F, S239T, S239H, S239Y, V240A, V240T,V240M, V263A, V263T, V263M, V264M, V264Y, V266A, V266T, V266M, E269H,E269Y, E269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V,A330I, A330F, A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H,L328D/I332E, L328E/I332E, L328N/I332E, L328Q/I332E, L328V/I332E,L328T/I332E, L328H/I332E, L328I/I332E, L328A, I332T, I332H, I332Y,I332A, S239E/V264I/I332E, S239Q/V264I/I332E, S239E/V264I/A330Y/I332E,S239E/V264I/S298A/A330Y/I332E, S239D/N297D/I332E, S239E/N297D/I332E,S239D/D265V/N297D/I332E, S239D/D265I/N297D/I332E,S239D/D265L/N297D/I332E, S239D/D265F/N297D/I332E,S239D/D265Y/N297D/I332E, S239D/D265H/N297D/I332E,S239D/D265T/N297D/I332E, V264E/N297D/I332E, Y296D/N297D/I332E,Y296E/N297D/I332E, Y296N/N297D/I332E, Y296Q/N297D/I332E,Y296H/N297D/I332E, Y296T/N297D/I332E, N297D/T299V/I332E,N297D/T299I/I332E, N297D/T299L/I332E, N297D/T299F/I332E,N297D/T299H/I332E, N297D/T299E/I332E, N297D/A330Y/I332E,N297D/S298A/A330Y/I332E, S239D/A330Y/I332E, S239N/A330Y/I332E,S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E,S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E,S239D/V264I/S298A/I332E, S239D/V264I/A330L/I332E, L328N, L328H,S239D/I332E/A330I, N297D/I332E/S239D/A330L, P230A, E233D, P230A/E233D,P230A/E233D/I332E, S267T, S267H, S267D, S267N, E269T, E269L, E269N,D270Q, D270T, D270H, E272S, E272K, E272I, E272Y, V273I, K274T, K274E,K274R, K274L, K274Y, F275W, N276S, N276E, N276R, N276L, N276Y, Y278T,Y278E, Y278K, Y278W, E283R, V302I, E318R, K320T, K320D, K320I, K322T,K322H, V323I, S324T, S324D, S324R, S324I, S324V, S324L, S324Y, K326L,K326I, K326T, A327D, A327T, A330S, A330W, A330M, P331V, P331H, E333T,E333H, E333I, E333Y, K334I, K334T, K334F, T335D, T335R, T335Y,L234I/L235D, V240I/V266I, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/V266I,S239D/A330Y/I332E/K326E, S239D/A330Y/I332E/K326T,S239D/N297D/I332E/A330Y,S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T,S239D/N297D/I332E/L235D, and S239D/N297D/I332E/K326E, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

It is a further object of the present invention to provide Fc variantsthat are selected from the group consisting of D221K, D221Y, K222E,K222Y, T223E, T223K, H224E, H224Y, T225E, T225, T225K, T225W, P227E,P227K, P227Y, P227G, P228E, P228K, P228Y, P228G, P230E, P230Y, P230G,A231E, A231K, A231Y, A231P, A231G, P232E, P232K, P232Y, P232G, E233N,E233Q, E233K, E233R, E233S, E233T, E233H, E233A, E233V, E233L, E233I,E233F, E233M, E233Y, E233W, E233G, L234K, L234R, L234S, L234A, L234M,L234W, L234P, L234G, L235E, L235K, L235R, L235A, L235M, L235W, L235P,L235G, G236D, G236E, G236N, G236Q, G236K, G236R, G236S, G236T, G236H,G236A, G236V, G236L, G236I, G236F, G236M, G236Y, G236W, G236P, G237D,G237E, G237N, G237Q, G237K, G237R, G237S, G237T, G237H, G237V, G237L,G237I, G237F, G237M, G237Y, G237W, G237P, P238D, P238E, P238N, P238Q,P238K, P238R, P238S, P238T, P238H, P238V, P238L, P238I, P238F, P238M,P238Y, P238W, P238G, S239Q, S239K, S239R, S239V, S239L, S239I, S239M,S239W, S239P, S239G, F241D, F241E, F241Y, F243E, K246D, K246E, K246H,K246Y, D249Q, D249H, D249Y, R255E, R255Y, E258S, E258H, E258Y, T260D,T260E, T260H, T260Y, V262E, V262F, V264D, V264E, V264N, V264Q, V264K,V264R, V264S, V264H, V264W, V264P, V264G, D265Q, D265K, D265R, D265S,D265T, D265H, D265V, D265L, D265I, D265F, D265M, D265Y, D265W, D265P,S267E, S267Q, S267K, S267R, S267V, S267L, S267I, S267F, S267M, S267Y,S267W, S267P, H268D, H268E, H268Q, H268K, H268R, H268T, H268V, H268L,H268I, H268F, H268M, H268W, H268P, H268G, E269K, E269S, E269V, E269I,E269M, E269W, E269P, E269G, D270R, D270S, D270L, D270I, D270F, D270M,D270Y, D270W, D270P, D270G, P271D, P271E, P271N, P271Q, P271K, P271R,P271S, P271T, P271H, P271A, P271V, P271L, P271I, P271F, P271M, P271Y,P271W, P271G, E272D, E272R, E272T, E272H, E272V, E272L, E272F, E272M,E272W, E272P, E272G, K274D, K274N, K274S, K274H, K274V, K274I, K274F,K274M, K274W, K274P, K274G, F275L, N276D, N276T, N276H, N276V, N276I,N276F, N276M, N276W, N276P, N276G, Y278D, Y278N, Y278Q, Y278R, Y278S,Y278H, Y278V, Y278L, Y278I, Y278M, Y278P, Y278G, D280K, D280L, D280W,D280P, D280G, G281D, G281K, G281Y, G281P, V282E, V282K, V282Y, V282P,V282G, E283K, E283H, E283L, E283Y, E283P, E283G, V284E, V284N, V284T,V284L, V284Y, H285D, H285E, H285Q, H285K, H285Y, H285W, N286E, N286Y,N286P, N286G, K288D, K288E, K288Y, K290D, K290N, K290H, K290L, K290W,P291D, P291E, P291Q, P291T, P291H, P291I, P291G, R292D, R292E, R292T,R292Y, E293N, E293R, E293S, E293T, E293H, E293V, E293L, E293I, E293F,E293M, E293Y, E293W, E293P, E293G, E294K, E294R, E294S, E294T, E294H,E294V, E294L, E294I, E294F, E294M, E294Y, E294W, E294P, E294G, Q295D,Q295E, Q295N, Q295R, Q295S, Q295T, Q295H, Q295V, Q295I, Q295F, Q295M,Q295Y, Q295W, Q295P, Q295G, Y296K, Y296R, Y296A, Y296V, Y296M, Y296G,N297Q, N297K, N297R, N297T, N297H, N297V, N297L, N297I, N297F, N297M,N297Y, N297W, N297P, N297G, S298D, S298E, S298Q, S298K, S298R, S298I,S298F, S298M, S298Y, S298W, T299D, T299E, T299N, T299Q, T299K, T299R,T299L, T299F, T299M, T299Y, T299W, T299P, T299G, Y300D, Y300E, Y300N,Y300Q, Y300K, Y300R, Y300S, Y300T, Y300H, Y300A, Y300V, Y300M, Y300W,Y300P, Y300G, R301D, R301E, R301H, R301Y, V303D, V303E, V303Y, S304D,S304N, S304T, S304H, S304L, V305E, V305T, V305Y, K317E, K317Q, E318Q,E318H, E318L, E318Y, K320N, K320S, K320H, K320V, K320L, K320F, K320Y,K320W, K320P, K320G, K322D, K322S, K322V, K322I, K322F, K322Y, K322W,K322P, K322G, S324H, S324F, S324M, S324W, S324P, S324G, N325K, N325R,N325S, N325F, N325M, N325Y, N325W, N325P, N325G, K326P, A327E, A327K,A327R, A327H, A327V, A327I, A327F, A327M, A327Y, A327W, A327P, L328D,L328Q, L328K, L328R, L328S, L328T, L328V, L328I, L328Y, L328W, L328P,L328G, P329D, P329E, P329N, P329Q, P329K, P329R, P329S, P329T, P329H,P329V, P329L, P329I, P329M, P329Y, P329W, P329G, A330E, A330N, A330T,A330P, A330G, P331D, P331Q, P331R, P331T, P331L, P331I, P331F, P331M,P331Y, P331W, I332K, I332R, I332S, I332V, I332L, I332F, I332M, I332W,I332P, I332G, E333L, E333F, E333M, E333P, K334P, T335N, T335S, T335H,T335V, T335L, T335I, T335F, T335M, T335W, T335P, T335G, I336E, I336K,I336Y, S337E, S337N, and S337H, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat.

It is a further object of the present invention to provide an Fc variantthat binds with greater affinity to one or more FcγRs. In oneembodiment, said Fc variants have affinity for an FcγR that is more than1-fold greater than that of the parent Fc polypeptide. In an alternateembodiment, said Fc variants have affinity for an FcγR that is more than5-fold greater than that of the parent Fc polypeptide. In a preferredembodiment, said Fc variants have affinity for an FcγR that is between5-fold and 300-fold greater than that of the parent Fc polypeptide. Inone embodiment, said Fc variants comprise at least one amino acidsubstitution at a position selected from the group consisting of: 230,233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302,318, 324, 325, 326, 328, 330, 332, and 335, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. In apreferred embodiment, said Fc variants comprise at least one amino acidsubstitution selected from the group consisting of: P230A, E233D, L234E,L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q,S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, 272Y, K274T,K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V,L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R,and T335Y, wherein the numbering of the residues in the Fc region isthat of the EU index as in Kabat. In a mostly preferred embodiment, saidFc variants are selected from the group consisting of V264I,F243L/V264I, L328M, I332E, L328M/I332E, V264I/I332E, S298A/I332E,S239E/I332E, S239Q/I332E, S239E, A330Y, I332D, L328I/I332E, L328Q/I332E,V264T, V240I, V266I, S239D, S239D/I332D, S239D/I332E, S239D/I332N,S239D/I332Q, S239E/I332D, S239E/I332N, S239E/I332Q, S239N/I332D,S239N/I332E, S239Q/I332D, A330Y/I332E, V264I/A330Y/I332E, A330L/I332E,V264I/A330L/I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I,S239T, V240M, V264Y, A330I, N325T, L328D/I332E, L328V/I332E,L328T/I332E, L328I/I332E, S239E/V264I/I332E, S239Q/V264I/I332E,S239E/V264I/A330Y/I332E, S239D/A330Y/I332E, S239N/A330Y/I332E,S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E,S239D/S298A/I332E, S239N/S298A/V332E, S239D/V264I/I332E,S239D/V264I/S298A/I332E, S239D/V264I/A330L/I332E, S239D/I332E/A330I,P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R, K274L, K274Y,F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T,T335D, T335R, T335Y, V240I/V266I, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, andS239D/A330Y/I332E/K326T, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat.

It is a further object of the present invention to provide Fc variantthat have a FcγRIIIa-fold:FcγRIIb-fold ratio greater than 1:1. In oneembodiment, said Fc variants have a FcγIIIa-fold:FcγRIIb-fold ratiogreater than 11:1. In a preferred embodiment, said Fc variants have aFcγRIIa-fold:FcγRIIb-fold ratio between 11:1 and 86:1. In oneembodiment, said Fc variants comprise at least one amino acidsubstitution at a position selected from the group consisting of: 234,235, 239, 240, 264, 296, 330, and I332, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. In apreferred embodiment, said Fc variants comprise at least one amino acidsubstitution selected from the group consisting of: L234Y, L234I, L235I,S239D, S239E, S239N, S239Q, V240A, V240M, V264I, V264Y, Y296Q, A330L,A330Y, A330I, I332D, and I332E, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat. In a mostly preferredembodiment, said Fc variants are selected from the group consisting of:I332E, V264I/I332E, S239E/I332E, S239Q/I332E, Y296Q, A330L, A330Y,I332D, S239D, S239D/I332E, A330Y/I332E, V264I/A330Y/I332E, A330L/I332E,V264I/A330L/I332E, L234Y, L234I, L235I, V240A, V240M, V264Y, A330I,S239D/A330L/I332E, S239D/S298A/I332E, S239N/S298A/I332E,S239D/V264I/I332E, S239D/V264I/S298A/I332E, and S239D/V264I/A330L/I332E,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

It is a further object of the present invention to provide Fc variantsthat mediate effector function more effectively in the presence ofeffector cells. In one embodiment, said Fc variants mediate ADCC that isgreater than that mediated by the parent Fc polypeptide. In a preferredembodiment, said Fc variants mediate ADCC that is more than 5-foldgreater than that mediated by the parent Fc polypeptide. In a mostlypreferred embodiment, said Fc variants mediate ADCC that is between5-fold and 1000-fold greater than that mediated by the parent Fcpolypeptide. In one embodiment, said Fc variants comprise at least oneamino acid substitution at a position selected from the group consistingof: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276,278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. In a preferred embodiment, said Fc variants comprise at least oneamino acid substitutions selected from the group consisting of: P230A,E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E,S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I,E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I,E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q,L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, 1332Q,T335D, T335R, and T335Y, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat. In a mostly preferredembodiment, said Fc variants are selected from the group consisting of:V264I, F243L/V264I, L328M, I332E, L328M/I332E, V264I/I332E, S298A/I332E,S239E/I332E, S239Q/I332E, S239E, A330Y, I332D, L328I/I332E, L328Q/I332E,V264T, V240I, V266I, S239D, S239D/I332D, S239D/I332E, S239D/I332N,S239D/I332Q, S239E/I332D, S239E/I332N, S239E/I332Q, S239N/I332D,S239N/I332E, S239Q/I332D, A330Y/I332E, V264I/A330Y/I332E, A330L/I332E,V264I/A330L/I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I,S239T, V240M, V264Y, A330I, N325T, L328D/I332E, L328V/I332E,L328T/I332E, L328I/I332E, S239E/V264I/I332E, S239Q/V264I/I332E,S239E/V264I/A330Y/I332E, S239D/A330Y/I332E, S239N/A330Y/I332E,S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E,S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E,S239D/V264I/S298A/I332E, S239D/V264I/A330L/I332E, S239D/I332E/A330I,P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R, K274L, K274Y,F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T,T335D, T335R, T335Y, V240I/V266I, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, andS239D/A330Y/I332E/K326T, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat.

It is a further object of the present invention to provide Fc variantsthat bind with weaker affinity to one or more FcγRs. In one embodiment,said Fc variants comprise at least one amino acid substitution at aposition selected from the group consisting of: 230, 233, 234, 235, 239,240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 270,273, 276, 278, 283, 296, 297, 298, 299, 313, 323, 324, 325, 327, 328,329, 330, 332, and 333, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat. In a preferred embodiment,said Fc variants comprise an amino acid substitution at a positionselected from the group consisting of: P230A, E233D, L234D, L234N,L234Q, L234T, L234H, L234V, L234F, L234I, L235N, L235Q, L235T, L235H,L235V, L235F, L235D, S239E, S239N, S239Q, S239F, S239H, S239Y, V240A,V240T, F241W, F241L, F241Y, F241E, F241R, F243W, F243L F243Y, F243R,F243Q, P244H, P245A, P247V, P247G, V262I, V262A, V262T, V262E, V263I,V263A, V263T, V263M, V264L, V264I, V264W, V264T, V264R, V264F, V264M,V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L, D265H,D265T, V266A, V266T, V266M, S267Q, S267L, E269H, E269Y, E269F, E269R,E269T, E269L, E269N, D270Q, D270T, D270H, V273I, N276S, N276E, N276R,N276Y, Y278E, Y278W, E283R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T,Y296L, Y296I, Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A,T299S, T299V, T299H, T299F, T299E, W313F, V323I, S324R, S324L, S324Y,N325Q, N325L, N325I, N325D, N325E, N325A, N325V, N325H, A327N, A327L,L328M, 328E, L328N, L328Q, A327D, A327T, L328F, L328H, L328A, L328N,L328H, P329F, A330L, A330V, A330F, A330R, A330H, I332N, I332Q, I332T,I332H, I332Y, I332A, E333T, and E333H, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. In amostly preferred embodiment, said Fc variants are selected from thegroup consisting of: V264L, F241W, F241L, F243W, F243L,F241L/F243L/V262I/V264I, F241W/F243W, F241W/F243W/V262A/V264A,F241L/V262I, F243L/V262I/L264W, F241Y/F243Y/V262T/V264T,F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E, L328F,P244H, P245A, P247V, W313F, P244H/P245A/P247V, P247G,F241E/F243R/V262E/V264R/I332E, F241E/F243Y/V262T/V264R/I332E, D265G,D265N, S239E/D265G, S239E/D265N, S239E/D265Q, Y296E, Y296Q, T299I,A327N, S267Q/A327S, S267L/A327S, A327L, P329F, A330L, N297S, N297D,N297S/I332E, I332N, I332Q, V264F, V263I, T299A, T299S, T299V, N325Q,N325L, N325I, S239N, S239F, S239N/I332N, S239N/I332Q, S239Q/I332N,S239Q/I332Q, Y296D, Y296N, L234D, L234N, L234Q, L234T, L234H, L234V,L234F, L235N, L235Q, L235T, L235H, L235V, L235F, S239H, S239Y, V240A,V263T, V263M, V264M, V266A, V266T, V266M, E269H, E269Y, E269F, E269R,Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330F, A330R, A330H,N325D, N325E, N325A, N325V, N325H, L328E/I332E, L328N/I332E,L328Q/I332E, L328H/I332E, L328A, I332T, I332H, I332Y, I332A, L328N,L328H, E233D, P230A/E233D, E269T, E269L, E269N, D270Q, D270T, D270H,V273I, N276S, N276E, N276R, N276Y, Y278E, Y278W, E283R, V323I, S324R,S324L, S324Y, A327D, A327T, E333T, E333H, and L234I/L235D, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

It is a further object of the present invention to provide Fc variantsthat mediate ADCC in the presence of effector cells less effectively. Inone embodiment, said Fc variants comprise at least one amino acidsubstitution at a position selected from the group consisting of: 230,233, 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265,266, 267, 269, 270, 273, 276, 278, 283, 296, 297, 298, 299, 313, 323,324, 325, 327, 328, 329, 330, 332, and 333, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. In apreferred embodiment, said Fc variants comprise at least one amino acidsubstitution at a position selected from the group consisting of: P230A,E233D, L234D, L234N, L234Q, L234T, L234H, L234V, L234F, L234I, L235N,L235Q, L235T, L235H, L235V, L235F, L235D, S239E, S239N, S239Q, S239F,S239H, S239Y, V240A, V240T, F241W, F241L, F241Y, F241E, F241R, F243W,F243L F243Y, F243R, F243Q, P244H, P245A, P247V, P247G, V262I, V262A,V262T, V262E, V263I, V263A, V263T, V263M, V264L, V264I, V264W, V264T,V264R, V264F, V264M, V264E, D265G, D265N, D265Q, D265Y, D265F, D265V,D265I, D265L, D265H, D265T, V266A, V266T, V266M, S267Q, S267L, E269H,E269Y, E269F, E269R, E269T, E269L, E269N, D270Q, D270T, D270H, V273I,N276S, N276E, N276R, N276Y, Y278E, Y278W, E283R, Y296E, Y296Q, Y296D,Y296N, Y296S, Y296T, Y296L, Y296I, Y296H, N297S, N297D, N297E, A298H,T299I, T299L, T299A, T299S, T299V, T299H, T299F, T299E, W313F, V323I,S324R, S324L, S324Y, N325Q, N325L, N325I, N325D, N325E, N325A, N325V,N325H, A327N, A327L, L328M, 328E, L328N, L328Q, A327D, A327T, L328F,L328H, L328A, L328N, L328H, P329F, A330L, A330V, A330F, A330R, A330H,I332N, I332Q, I332T, I332H, I332Y, I332A, E333T, and E333H, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. In a mostly preferred embodiment, said Fc variants are selectedfrom the group consisting of: V264L, F241W, F241L, F243W, F243L,F241L/F243I/V262I/V264I, F241W/F243W, F241W/F243W/V262A/V264A,F241L/V262I, F243L/V262I/V264W, F241Y/F243Y/V262T/V264T,F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E, L328F,P244H, P245A, P247V, W313F, P244H/P245A/P247V, P247G,F241E/F243R/V262E/N264R/I332E, F241E/F243Y/V262T/V264R/I332E, D265G,D265N, S239E/D265G, S239E/D265N, S239E/D265Q, Y296E, Y296Q, T299I,A327N, S267Q/A327S, S267L/A327S, A327L, P329F, A330L, N297S, N297D,N297S/I332E, I332N, I332Q, V264F, V263I, T299A, T299S, T299V, N325Q, N325L, N325I, S239N, S239F, S239N/I332N, S239N/I332Q, S239Q/I332N,S239Q/I332Q, Y296D, Y296N, L234D, L234N, L234Q, L234T, L234H, L234V,L234F, L235N, L235Q, L235T, L235H, L235V, L235F, S239H, S239Y, V240A,V263T, V263M, V264M, V266A, V266T, V266M, E269H, E269Y, E269F, E269R,Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330F, A330R, A330H,N325D, N325E, N325A, N325V, N325H, L328E/I332E, L328N/I332E,L328Q/I332E, L328H/I332E, L328A, I332T, I332H, I332Y, I332A, L328N,L328H, E233D, P230A/E233D, E269T, E269L, E269N, D270Q, D270T, D270H,V273I, N276S, N276E, N276R, N276Y, Y278E, Y278W, E283R, V323I, S324R,S324L, S324Y, A327D, A327T, E333T, E333H, and L234I/L235D, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

It is a further object of the present invention to provide Fc variantsthat have improved function and/or solution properties as compared tothe aglycosylated form of the parent Fc polypeptide. Improvedfunctionality herein includes but is not limited to binding affinity toan Fc ligand. Improved solution properties herein includes but is notlimited to stability and solubility. In one embodiment, saidaglycosylated Fc variants bind to an FcγR with an affinity that iscomparable to or better than the glycosylated parent Fc polypeptide. Inan alternate embodiment, said Fc variants bind to an FcγR with anaffinity that is within 0.4-fold of the glycosylated form of the parentFc polypeptide. In one embodiment, said Fc variants comprise at leastone amino acid substitution at a position selected from the groupconsisting of: 239, 241, 243, 262, 264, 265, 296, 297, 330, and 332,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat. In a preferred embodiment, said Fc variants comprisean amino acid substitution selected from the group consisting of: S239D,S239E, F241Y, F243Y, V262T, V264T, V264E, D265Y, D265H, D265V, D265I,Y296N, N297D, A330Y, and I332E, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat. In a mostly preferredembodiment, said Fc variants are selected from the group consisting of:N297D/I332E, F241Y/F243Y/V262T/V264T/N297D/I332E, S239D/N297D/I332E,S239E/N297D/I332E, S239D/D265Y/N297D/I332E, S239D/D265H/N297D/I332E,V264E/N297D/I332E, Y296N/N297D/I332E, N297D/A330Y/I332E,S239D/D265V/N297D/I332E, S239D/D265I/N297D/I332E, andN297D/S298A/A330Y/I332E, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat.

The present invention also provides methods for engineering optimized Fcvariants. It is an object of the present invention to provide designstrategies that may be used to guide Fc optimization. It is a furtherobject of the present invention to provide computational screeningmethods that may be used to design Fc variants. It is a further objectof the present invention to provide methods for generating libraries forexperimental testing. It is a further object of the present invention toprovide experimental production and screening methods for obtainingoptimized Fc variants.

The present invention provides isolated nucleic acids encoding the Fcvariants described herein. The present invention provides vectorscomprising said nucleic acids, optionally, operably linked to controlsequences. The present invention provides host cells containing thevectors, and methods for producing and optionally recovering the Fcvariants.

The present invention provides novel antibodies and Fc fusions thatcomprise the Fc variants disclosed herein. Said novel antibodies and Fcfusions may find use in a therapeutic product.

The present invention provides compositions comprising antibodies and Fcfusions that comprise the Fc variants described herein, and aphysiologically or pharmaceutically acceptable carrier or diluent.

The present invention contemplates therapeutic and diagnostic uses forantibodies and Fc fusions that comprise the Fc variants disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Antibody structure and function. Shown is a model of a fulllength human IgG1 antibody, modeled using a humanized Fab structure frompdb accession code 1CE1 (James et al., 1999, J Mol Biol 289:293-301) anda human IgG1 Fc structure from pdb accession code 1D/V2 (DeLano et al.,2000, Science 287:1279-1283). The flexible hinge that links the Fab andFc regions is not shown. IgG1 is a homodimer of heterodimers, made up oftwo light chains and two heavy chains. The Ig domains that comprise theantibody are labeled, and include V_(L) and C_(L) for the light chain,and V_(H), Cgamma1 (Cγ1), Cgamma2 (Cγ2), and Cgamma3 (Cγ3) for the heavychain. The Fc region is labeled. Binding sites for relevant proteins arelabeled, including the antigen binding site in the variable region, andthe binding sites for FcγRs, FcRn, C1q, and proteins A and G in the Fcregion.

FIG. 2. The Fc/FcγRIIIb complex structure 1IIS. Fc is shown as a grayribbon diagram, and FcγRIIb is shown as a black ribbon. The N297carbohydrate is shown as black sticks.

FIG. 3. The amino acid sequence of the heavy chain of the antibodyalemtuzumab (Campath®, a registered trademark of llex PharmaceuticalsLP), illustrating positions numbered sequentially (2 lines above theamino acid sequence) and positions numbered according to the EU index asin Kabat (2 lines below the amino acid sequence. The approximatebeginnings of Ig domains VH1, Cγ1, the hinge, Cγ2, and Cγ3 are alsolabeled above the sequential numbering. Polymorphisms have been observedat a number of Fc positions, including but not limited to Kabat 270,272, 312, 315, 356, and 358, and thus slight differences between thepresented sequence and sequences in the prior art may exist.

FIG. 4. Experimental library residues mapped onto the Fc/FcγRIIIbcomplex structure 1IIS. Fc is shown as a gray ribbon diagram, andFcγRIIIb is shown as a black ribbon. Experimental library residues areshown in black, the N297 carbohydrate is shown in grey.

FIG. 5. The human IgG1 Fc sequence showing positions relevant to thedesign of the Fc variant experimental library. The sequence includes thehinge region, domain Cγ2, and domain Cγ3. Residue numbers are accordingto the EU index as in Kabat. Positions relevant to the experimentallibrary are underlined. Because of observed polymorphic mutations at anumber of Fc positions, slight differences between the presentedsequence and sequences in the literature may exist.

FIG. 6. Expression of Fc variant and wild type (WT) proteins ofalemtuzumab in 293T cells. Plasmids containing alemtuzumab heavy chaingenes (WT or variants) were co-transfected with plasmid containing thealemtuzumab light chain gene. Media were harvested 5 days aftertransfection. For each transfected sample, 10 ul medium was loaded on aSDS-PAGE gel for Western analysis. The probe for Western wasperoxidase-conjugated goat-anti human IgG (Jackson Immuno-Research,catalog #109-035-088). WT: wild type alemtuzumab; 1-10: alemtuzumabvariants. H and L indicate antibody heavy chain and light chain,respectively.

FIG. 7. Purification of alemtuzumab using protein A chromatography. WTalemtuzumab proteins was expressed in 293T cells and the media washarvested 5 days after transfection. The media were diluted 1:1 with PBSand purified with protein A (Pierce, Catalog #20334). O: original samplebefore purification; FT: flow through; E: elution; C: concentrated finalsample. The left picture shows a Simple Blue-stained SDS-PAGE gel, andthe right shows a western blot labeled using peroxidase-conjugatedgoat-anti human IgG.

FIG. 8. Production of deglycosylated antibodies. Wild type and variantsof alemtuzumab were expressed in 293T cells and purified with protein Achromatography. Antibodies were incubated with peptide-N-glycosidase(PNGase F) at 37° C. for 24 h. For each antibody, a mock treated sample(-PNGase F) was done in parallel. WT: wild-type alemtuzumab; #15, #16,#17, #18, #22: alemtuzumab variants F241E/F243R/V262E/V264R,F241E/F243Q/V262T/V264E, F241R/F243Q/V262T/V264R,F241E/F243Y/V262T/V264R, and I332E respectively. The faster migration ofthe PNGase F treated versus the mock treated samples represents thedeglycosylated heavy chains.

FIG. 9. Alemtuzumab expressed from 293T cells binds its antigen. Theantigenic CD52 peptide, fused to GST, was expressed in E. coli BL21(DE3) under IPTG induction. Both uninduced and induced samples were runon a SDS-PAGE gel, and transferred to PVDF membrane. For westernanalysis, either alemtuzumab from Sotec (α-CD52, Sotec) (finalconcentration 2.5 ng/ul) or media of transfected 293T cells (Campath,Xencor) (final alemtuzumab concentration approximately 0.1-0.2 ng/ul)were used as primary antibody, and peroxidase-conjugated goat-anti humanIgG was used as secondary antibody. M: pre-stained marker; U: un-inducedsample for GST-CD52; I: induced sample for GST-CD52.

FIG. 10. Expression and purification of extracellular region of humanV158 FcγRIIIa. Tagged FcγRIIIa was transfected in 293T cells, and mediacontaining secreted FcγRIIIa were harvested 3 days later and purifiedusing affinity chromatography. 1: media; 2: flow through; 3: wash; 4-8:serial elutions. Both simple blue-stained SDS-PAGE gel and westernresult are shown. For the western blot, membrane was probed withanti-GST antibody.

FIG. 11. Binding to human V158 FcγRIIIa by select alemtuzumab Fcvariants from the experimental library as determined by the AlphaScreen™assay, described in Example 2. In the presence of competitor antibody(Fc variant or WT alemtuzumab) a characteristic inhibition curve isobserved as a decrease in luminescence signal. Phosphate buffer saline(PBS) alone was used as the negative control. The binding data werenormalized to the maximum and minimum luminescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a one site competition model using nonlinear regression. These fitsprovide IC50s for each antibody, illustrated for WT and S239D by thedotted lines.

FIGS. 12. AlphaScreen™ assay showing binding of select alemtuzumab Fcvariants to human FcγRIIb. The binding data were normalized to the upperand lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIGS. 13 a and 13 b. AlphaScreen™ assay showing binding of selectalemtuzumab (FIG. 13 a) and trastuzumab (FIG. 13 b) Fc variants to humanVal158 FcγRIIIa. The binding data were normalized to the upper and lowerbaselines for each particular antibody, and the curves represent thefits of the data to a one site competition model. PBS was used as anegative control.

FIGS. 14 a and 14 b. AlphaScreen™ assay measuring binding to human V158FcγRIIIa by select Fc variants in the context of trastuzumab. Thebinding data were normalized to the upper and lower baselines for eachparticular antibody, and the curves represent the fits of the data to aone site competition model. PBS was used as a negative control.

FIGS. 15 a and 15 b. AlphaScreen™ assay measuring binding to human V158FcγRIIIa by select Fc variants in the context of rituximab (FIG. 15 a)and cetuximab (FIG. 15 b). The binding data were normalized to the upperand lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIGS. 16 a-16 b. AlphaScreen™ assay comparing binding of selectalemtuzumab Fc variants to human V158 FcγRIIIa (FIG. 16 a) and humanFcγRIIb (FIG. 16 b). The binding data were normalized to the upper andlower baselines for each particular antibody, and the curves representthe fits of the data to a one site competition model. PBS was used as anegative control.

FIG. 17. AlphaScreen™ assay measuring binding to human V158 FcγRIIIa byselect Fc variants in the context of trastuzumab. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIG. 18. AlphaScreen™ assay showing binding of select alemtuzumab Fcvariants to human R131 FcγRIIa. The binding data were normalized to theupper and lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model.

FIGS. 19 a and 19 b. AlphaScreen™ assay showing binding of selectalemtuzumab Fc variants to human V158 FcγRIIIa. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIG. 20. AlphaScreen™ assay showing binding of aglycosylated alemtuzumabFc variants to human V158 FcγRIIIa. The binding data were normalized tothe upper and lower baselines for each particular antibody, and thecurves represent the fits of the data to a one site competition model.PBS was used as a negative control.

FIG. 21. AlphaScreen™ assay comparing human V158 FcγRIIIa binding byselect alemtuzumab Fc variants in glycosylated (solid symbols, solidlines) and deglycosylated (open symbols, dotted lines). The binding datawere normalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model.

FIGS. 22 a-22 b. AlphaScreen™ assay showing binding of selectalemtuzumab Fc variants to the V158 (FIG. 22 a) and F158 (FIG. 22 b)allotypes of human FcγRIIIa. The binding data were normalized to theupper and lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIGS. 23 a-23 d. FIGS. 23 a and 23 b show the correlation between SPRKd's and AlphaScreen™ IC50's from binding of select alemtuzumab Fcvariants to V158 FcγIIIa (FIG. 23 a) and F158 FcγRIIIa (FIG. 23 b).FIGS. 23 c and 23 d show the correlation between SPR and AlphaScreenfold-improvements over WT for binding of select alemtuzumab Fc variantsto V158 FcγRIIIa (FIG. 23 c) and F158 FcγRIIIa (FIG. 23 d). Binding dataare presented in Table 63. The lines through the data represent thelinear fits of the data, and the r² values indicate the significance ofthese fits.

FIGS. 24 a-24 b. Cell-based ADCC assays of select Fc variants in thecontext of alemtuzumab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay (Perkin Elmer, MA), as described in Example 7, usingDoHH-2 lymphoma target cells and 50-fold excess human PBMCs. FIG. 24 ais a bar graph showing the raw fluorescence data for the indicatedalemtuzumab antibodies at 10 ng/ml. The PBMC bar indicates basal levelsof cytotoxicity in the absence of antibody. FIG. 24 b shows thedose-dependence of ADCC on antibody concentration for the indicatedalemtuzumab antibodies, normalized to the minimum and maximumfluorescence signal for each particular curve, provided by the baselinesat low and high antibody concentrations respectively. The curvesrepresent the fits of the data to a sigmoidal dose-response model usingnonlinear regression.

FIGS. 25 a-25 c. Cell-based ADCC assays of select Fc variants in thecontext of trastuzumab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay, as described in Example 7, using BT474 and Sk-Br-3breast carcinoma target cells and 50-fold excess human PBMCs. FIG. 25 ais a bar graph showing the raw fluorescence data for the indicatedtrastuzumab antibodies at 1 ng/ml. The PBMC bar indicates basal levelsof cytotoxicity in the absence of antibody. FIGS. 25 b and 25 c show thedose-dependence of ADCC on antibody concentration for the indicatedtrastuzumab antibodies, normalized to the minimum and maximumfluorescence signal for each particular curve, provided by the baselinesat low and high antibody concentrations respectively. The curvesrepresent the fits of the data to a sigmoidal dose-response model usingnonlinear regression.

FIGS. 26 a-26 c. Cell-based ADCC assays of select Fc variants in thecontext of rituximab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay, as described in Example 7, using WIL2-S lymphomatarget cells and 50-fold excess human PBMCs. FIG. 26 a is a bar graphshowing the raw fluorescence data for the indicated rituximab antibodiesat 1 ng/ml. The PBMC bar indicates basal levels of cytotoxicity in theabsence of antibody. FIGS. 26 b and 26c show the dose-dependence of ADCCon antibody concentration for the indicated rituximab antibodies,normalized to the minimum and maximum fluorescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a sigmoidal dose-response model using nonlinear regression.

FIGS. 27 a-27 b. Cell-based ADCC assay of select trastuzumab (FIG. 27 a)and rituximab (FIG. 27 b) Fc variants showing enhancements in potencyand efficacy. Both assays used homozygous F158/F158 FcγRIIIa PBMCs aseffector cells at a 25-fold excess to target cells, which were Sk-Br-3for the trastuzumab assay and WIL2-S for the rituximab assay. Data werenormalized according to the absolute minimal lysis for the assay,provided by the fluorescence signal of target cells in the presence ofPBMCs alone (no antibody), and the absolute maximal lysis for the assay,provided by the fluorescence signal of target cells in the presence ofTriton X1000, as described in Example 7.

FIG. 28. Cell-based ADCC assay of select trastuzumab Fc variants againstdifferent cell lines expressing varying levels of the Her2/neu targetantigen. ADCC assays were run as described in Example 7, with variouscell lines expressing amplified to low levels of Her2/neu receptor,including Sk-Br-3 (1×10⁶ copies), SkOV3 (˜1×10⁵), OVCAR3(˜1×10⁴), andMCF-7 (˜3×10³ copies). Human PBMCs allotyped as homozygous F158/F158FcγRIIIa were used at 25-fold excess to target cells. The bar graphprovides ADCC data for WT and Fc variant against the indicated celllines, normalized to the minimum and maximum fluorescence signalprovided by minimal lysis (PBMCs alone) and maximal lysis (TritonX1000).

FIG. 29. Cell-based ADCC assays of select Fc variants in the context oftrastuzumab using natural killer (NK) cells as effector cells andmeasuring LDH release to monitor cell lysis. NK cells, allotyped asheterozygous V158/F158 FcγRIIIa, were at an 8-fold excess to Sk-Br-3breast carcinoma target cells, and the level of cytotoxicity wasmeasured using the LDH Cytotoxicity Detection Kit, according to themanufacturer's protocol (Roche Diagnostics GmbH, Penzberg, Germany). Thegraph shows the dose-dependence of ADCC on antibody concentration forthe indicated trastuzumab antibodies, normalized to the minimum andmaximum fluorescence signal for each particular curve, provided by thebaselines at low and high antibody concentrations respectively. Thecurves represent the fits of the data to a sigmoidal dose-response modelusing nonlinear regression.

FIG. 30. Cell-based ADCP assay of select variants. The ADCP assay wascarried out as described in Example 8, using a co-labeling strategycoupled with flow cytometry. Differentiated macrophages were used aseffector cells, and Sk-Br-3 cells were used as target cells. Percentphagocytosis represents the number of co-labeled cells(macrophage+Sk-Br-3) over the total number of Sk-Br-3 in the population(phagocytosed+non-phagocytosed).

FIGS. 31 a-31 c. Capacity of select Fc variants to mediate binding andactivation of complement. FIG. 31 a shows an AlphaScreen™ assaymeasuring binding of select alemtuzumab Fc variants to C1q. The bindingdata were normalized to the upper and lower baselines for eachparticular antibody, and the curves represent the fits of the data to aone site competition model. FIGS. 31 b and 31 c show a cell-based assaymeasuring capacity of select rituximab Fc variants to mediate CDC. CDCassays were performed using Alamar Blue to monitor lysis of Fc variantand WT rituximab -opsonized WIL2-S lymphoma cells by human serumcomplement (Quidel, San Diego, Calif.). The dose-dependence on antibodyconcentration of complement-mediated lysis is shown for the indicatedrituximab antibodies, normalized to the minimum and maximum fluorescencesignal for each particular curve, provided by the baselines at low andhigh antibody concentrations respectively. The curves represent the fitsof the data to a sigmoidal dose-response model using nonlinearregression.

FIG. 32. AlphaScreen™ assay measuring binding of select alemtuzumab Fcvariants to bacterial protein A, as described in Example 10. The bindingdata were normalized to the upper and lower baselines for eachparticular antibody, and the curves represent the fits of the data to aone site competition model. PBS was used as a negative control.

FIG. 33. AlphaScreen™ assay measuring binding of select alemtuzumab Fcvariants to human FcRn, as described in Example 10. The binding datawere normalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIGS. 34 a and 34 b. AlphaScreen™ assay measuring binding of selectalemtuzumab (FIG. 34 a) and trastuzumab (FIG. 34 b) Fc variants to mouseFcγRIII, as described in Example 11. The binding data were normalized tothe upper and lower baselines for each particular antibody, and thecurves represent the fits of the data to a one site competition model.PBS was used as a negative control.

FIG. 35. Cell-based ADCC assays of select Fc variants in the context oftrastuzumab using mouse PBMCs as effector cells. ADCC was measured usingthe DELFIA® EuTDA-based cytotoxicity assay using Sk-Br-3 breastcarcinoma target cells and 8-fold excess mouse PBMCs. The bar graphshows the raw fluorescence data for the indicated trastuzumab antibodiesat 10 ng/ml. The PBMC bar indicates basal levels of cytotoxicity in theabsence of antibody, and TX indicates complete cell lysis in thepresence of Triton X1000.

FIG. 36. AlphaScreen™ assay measuring binding to human V158 FcγRIIIa byselect trastuzumab Fc variants expressed in 293T and CHO cells, asdescribed in Example 12. The binding data were normalized to the upperand lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIGS. 37 a-37 b. Synergy of Fc variants and engineered glycoforms. FIG.37 a presents an AlphaScreen™ assay showing V158 FcγRIIIa binding by WTand Fc variant (V209, S239/1332E/A330L) trastuzumab expressed in 293T,CHO, and Lec-13 CHO cells. The data were normalized to the upper andlower baselines for each antibody, and the curves represent the fits ofthe data to a one site competition model. PBS was used as a negativecontrol. FIG. 37 b presents a cell-based ADCC assay showing the abilityof 239T, CHO, and Lec-13 CHO expressed WT and V209 trastuzumab tomediate ADCC. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay as described previously, with Sk-Br-3 breastcarcinoma target cells. The data show the dose-dependence of ADCC onantibody concentration for the indicated trastuzumab antibodies,normalized to the minimum and maximum fluorescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a sigmoidal dose-response model using nonlinear regression.

FIGS. 38 a-38 c. Sequences showing improved anti-CD20 antibodies. Thelight and heavy chain sequences of rituximab are presented in FIG. 38 aand FIG. 38 b respectively, and are taken from translated Sequence 3 ofU.S. Pat. No. 5,736,137. Relevant positions in FIG. 38 b are bolded,including S239, V240, V264I, E272, K274, N297, S298, K326, A330, andI332. FIG. 38 c shows the improved anti-CD20 antibody heavy chainsequences, with variable positions designated in bold as X₁, X₂, X₃, X₄,X₅, X₆, X₇, X₈, Z₁, and Z₂. The table below the sequence providespossible substitutions for these positions. The improved anti-CD20antibody sequences comprise at least one non-WT amino acid selected fromthe group of possible substitutions for X₁, X₂, X₃, X₄, X₅, X₆, X₇, andX₈. These improved anti-CD20 antibody sequences may also comprise asubstitution Z₁ and/or Z₂. These positions are numbered according to theEU index as in Kabat, and thus do not correspond to the sequential orderin the sequence.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocvtosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. The preferredamino acid modification herein is a substitution. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution I332E refers to avariant polypeptide, in this case an Fc variant, in which the isoleucineat position 332 is replaced with a glutamic acid.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (μ), delta (δ), gamma (γ), sigma (ε), and alpha(α) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively.Antibody herein is meant to include full length antibodies and antibodyfragments, and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.The term “antibody” includes antibody fragments, as are known in theart, such as Fab, Fab′, F(ab′)₂, Fv, scFv, or other antigen-bindingsubsequences of antibodies, either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAtechnologies. Particularly preferred are full length antibodies thatcomprise Fc variants as described herein. The term “antibody” comprisesmonoclonal and polyclonal antibodies. Antibodies can be antagonists,agonists, neutralizing, inhibitory, or stimulatory.

The antibodies of the present invention may be nonhuman, chimeric,humanized, or fully human. For a description of the concepts of chimericand humanized antibodies see Clark et al., 2000 and references citedtherein (Clark, 2000, Immunol Today 21:397-402). Chimeric antibodiescomprise the variable region of a nonhuman antibody, for example VH andVL domains of mouse or rat origin, operably linked to the constantregion of a human antibody (see for example U.S. Pat. No. 4,816,567). Ina preferred embodiment, the antibodies of the present invention arehumanized. By “humanized” antibody as used herein is meant an antibodycomprising a human framework region (FR) and one or more complementaritydetermining regions (CDR's) from a non-human (usually mouse or rat)antibody. The non-human antibody providing the CDR's is called the“donor” and the human immunoglobulin providing the framework is calledthe “acceptor”. Humanization relies principally on the grafting of donorCDRs onto acceptor (human) VL and VH frameworks (Winter U.S. Pat. No.5,225,539). This strategy is referred to as “CDR grafting”.“Backmutation” of selected acceptor framework residues to thecorresponding donor residues is often required to regain affinity thatis lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337;6,054,297; 6,407,213). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Methods for humanizing non-human antibodiesare well known in the art, and can be essentially performed followingthe method of Winter and co-workers (Jones et al., 1986, Nature321:522-525; Riechmann et al.,1988, Nature 332:323-329; Verhoeyen etal., 1988, Science, 239:1534-1536). Additional examples of humanizedmurine monoclonal antibodies are also known in the art, for exampleantibodies binding human protein C (O'Connor et al., 1998, Protein Eng11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl AcadSci, USA 86:10029-33), and human epidermal growth factor receptor 2(Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9). In an alternateembodiment, the antibodies of the present invention may be fully human,that is the sequences of the antibodies are completely or substantiallyhuman. A number of methods are known in the art for generating fullyhuman antibodies, including the use of transgenic mice (Bruggemann etal., 1997, Curr Opin Biotechnol 8:455-458) or human antibody librariescoupled with selection methods (Griffiths et al., 1998, Curr OpinBiotechnol 9:102-108).

Specifically included within the definition of “antibody” areaglycosylated antibodies. By “aglycosvlated antibody” as used herein ismeant an antibody that lacks carbohydrate attached at position 297 ofthe Fc region, wherein numbering is according to the EU system as inKabat. The aglycosylated antibody may be a deglycosylated antibody, thatis an antibody for which the Fc carbohydrate has been removed, forexample chemically or enzymatically. Alternatively, the aglycosylatedantibody may be a nonglycosylated or unglycosylated antibody, that is anantibody that was expressed without Fc carbohydrate, for example bymutation of one or residues that encode the glycosylation pattern or byexpression in an organism that does not attach carbohydrates toproteins, for example bacteria.

Specifically included within the definition of “antibody” arefull-length antibodies that contain an Fc variant portion. By “fulllength antibody” herein is meant the structure that constitutes thenatural biological form of an antibody, including variable and constantregions. For example, in most mammals, including humans and mice, thefull length antibody of the IgG class is a tetramer and consists of twoidentical pairs of two immunoglobulin chains, each pair having one lightand one heavy chain, each light chain comprising immunoglobulin domainsV_(L) and C_(L), and each heavy chain comprising immunoglobulin domainsV_(H), Cγ1, Cγ2, and Cγ3. In some mammals, for example in camels andllamas, IgG antibodies may consist of only two heavy chains, each heavychain comprising a variable domain attached to the Fc region. By “IgG”as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. By “protein” herein ismeant at least two covalently attached amino acids, which includesproteins, polypeptides, oligopeptides and peptides. The protein may bemade up of naturally occurring amino acids and peptide bonds, orsynthetic peptidomimetic structures, i.e. “analogs”, such as peptoids(see Simon et al., 1992, Proc Natl Acad Sci USA 89(20):9367)particularly when LC peptides are to be administered to a patient. Thus“amino acid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homophenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chain may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation.

By “computational screening method” herein is meant any method fordesigning one or more mutations in a protein, wherein said methodutilizes a computer to evaluate the energies of the interactions ofpotential amino acid side chain substitutions with each other and/orwith the rest of the protein. As will be appreciated by those skilled inthe art, evaluation of energies, referred to as energy calculation,refers to some method of scoring one or more amino acid modifications.Said method may involve a physical or chemical energy term, or mayinvolve knowledge-, statistical-, sequence-based energy terms, and thelike. The calculations that compose a computational screening method areherein referred to as “computational screening calculations”.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γγ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys. By “library” herein is meant a set of Fc variantsin any form, including but not limited to a list of nucleic acid oramino acid sequences, a list of nucleic acid or amino acid substitutionsat variable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising the Fcvariant proteins, either in purified or unpurified form.

By “Fc”, “Fc region”, FC polypeptide”, etc. as used herein is meant anantibody as defined herein that includes the polypeptides comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, and the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM Fc may include the J chain.For IgG, as illustrated in FIG. 1, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Fc may refer to this region inisolation, or this region in the context of an antibody, antibodyfragment, or Fc fusion. An Fc may be an antibody, Fc fusion, or anprotein or protein domain that comprises Fc. Particularly preferred areFc variants, which are non-naturally occurring variants of an Fc.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to an Fc region or a derivative thereof.Fc fusion is herein meant to be synonymous with the terms“immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin”(sometimes with dashes) as used in the prior art (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general can be any protein or smallmolecule. The role of the non-Fc part of an Fc fusion, i.e. the fusionpartner, is to mediate target binding, and thus it is functionallyanalogous to the variable regions of an antibody. Virtually any proteinor small molecule may be linked to Fc to generate an Fc fusion. Proteinfusion partners may include, but are not limited to, the target-bindingregion of a receptor, an adhesion molecule, a ligand, an enzyme, acytokine, a chemokine, or some other protein or protein domain. Smallmolecule fusion partners may include any therapeutic agent that directsthe Fc fusion to a therapeutic target. Such targets may be any molecule,preferrably an extracellular receptor, that is implicated in disease.Two families of surface receptors that are targets of a number ofapproved small molecule drugs are G-Protein Coupled Receptors (GPCRs),and ion channels, including K+, Na+, Ca+ channels. Nearly 70% of alldrugs currently marketed worldwide target GPCRs. Thus the Fc variants ofthe present invention may be fused to a small molecule that targets, forexample, one or more GABA receptors, purinergic receptors, adrenergicreceptors, histaminergic receptors, opiod receptors, chemokinereceptors, glutamate receptors, nicotinic receptors, the 5HT (serotonin)receptor, and estrogen receptors. A fusion partner may be asmall-molecule mimetic of a protein that targets a therapeuticallyuseful target. Specific examples of particular drugs that may serve asFc fusion partners can be found in L. S. Goodman et al., Eds., Goodmanand Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill, NewYork, ed. 9, 1996). Fusion partners include not only small molecules andproteins that bind known targets for existing drugs, but orphanreceptors that do not yet exist as drug targets. The completion of thegenome and proteome projects are proving to be a driving force in drugdiscovery, and these projects have yielded a trove of orphan receptors.There is enormous potential to validate these new molecules as drugtargets, and develop protein and small molecule therapeutics that targetthem. Such protein and small molecule therapeutics are contemplated asFc fusion partners that employ the Fc variants of the present invention.A variety of linkers, defined and described below, may be used tocovalently link Fc to a fusion partner to generate an Fc fusion.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65), aswell as any undiscovered human FcγRs or FcγR isoforms or allotypes. AnFcγR may be from any organism, including but not limited to humans,mice, rats, rabbits, and monkeys. Mouse FcγRs include but are notlimited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms orallotypes.

By “Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136). Fc ligands may includeundiscovered molecules that bind Fc.

By “LqG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoglobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic □-sandwich folding topology. Theknown Ig domains in the IgG class of antibodies are V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “parent polypeptide” or “precursor polypeptide” (including Fc parentor precursors) as used herein is meant a polypeptide that issubsequently modified to generate a variant. Said parent polypeptide maybe a naturally occurring polypeptide, or a variant or engineered versionof a naturally occurring polypeptide. Parent polypeptide may refer tothe polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant an unmodified Fcpolypeptide that is modified to generate a variant, and by “parentantibody” as used herein is meant an unmodified antibody that ismodified to generate a variant antibody.

As outlined above, certain positions of the Fc molecule can be altered.By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. For example,position 297 is a position in the human antibody IgG1. Correspondingpositions are determined as outlined above, generally through alignmentwith other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or V_(H) genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide” as used herein is meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification. Variant polypeptide may refer to thepolypeptide itself, a composition comprising the polypeptide, or theamino sequence that encodes it. Preferably, the variant polypeptide hasat least one amino acid modification compared to the parent polypeptide,e.g. from about one to about ten amino acid modifications, andpreferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent polypeptidesequence, and most preferably at least about 90% homology, morepreferably at least about 95% homology. Accordingly, by “Fc variant” asused herein is meant an Fc sequence that differs from that of a parentFc sequence by virtue of at least one amino acid modification. An Fcvariant may only encompass an Fc region, or may exist in the context ofan antibody, Fc fusion, or other polypeptide that is substantiallyencoded by Fc. Fc variant may refer to the Fc polypeptide itself,compositions comprising the Fc variant polypeptide, or the amino acidsequence that encodes it. In a preferred embodiment, the variantproteins of the invention comprise an Fc variant, as described herein,and as such, may comprise an antibody (and the correspondingderivatives) with the Fc variant, or an Fc fusion protein that comprisesthe Fc variant. In addition, in some cases, the Fc is a variant ascompared to a wild-type Fc, or to a “parent” variant.

For all positions discussed in the present invention, numbering of animmunoglobulin heavy chain is according to the EU index (Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed., UnitedStates Public Health Svice, National Institutes of Health, Bethesda).The “EU index as in Kabat” refers to the residue numbering of the humanIgG1 EU antibody.

The Fc variants of the present invention may be optimized for a varietyof properties. Properties that may be optimized include but are notlimited to enhanced or reduced affinity for an FcγR. In a preferredembodiment, the Fc variants of the present invention are optimized topossess enhanced affinity for a human activating FcγR, preferably FcγRI,FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, most preferably FcγRIIIa. Inan alternately preferred embodiment, the Fc variants are optimized topossess reduced affinity for the human inhibitory receptor FcγRIIb.These preferred embodiments are anticipated to provide antibodies and Fcfusions with enhanced therapeutic properties in humans, for exampleenhanced effector function and greater anti-cancer potency. In analternate embodiment, the Fc variants of the present invention areoptimized to have reduced or ablated affinity for a human FcγR,including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa,and FcγRIIIb. These embodiments are anticipated to provide antibodiesand Fc fusions with enhanced therapeutic properties in humans, forexample reduced effector function and reduced toxicity. Preferredembodiments comprise optimization of Fc binding to a human FcγR, howeverin alternate embodiments the Fc variants of the present inventionpossess enhanced or reduced affinity for FcγRs from nonhuman organisms,including but not limited to mice, rats, rabbits, and monkeys. Fcvariants that are optimized for binding to a nonhuman FcγR may find usein experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies or Fc fusions that comprise Fc variants that are optimizedfor one or more mouse FcγRs, may provide valuable information withregard to the efficacy of the antibody or Fc fusion, its mechanism ofaction, and the like. The Fc variants of the present invention may alsobe optimized for enhanced functionality and/or solution properties inaglycosylated form. In a preferred embodiment, the aglycosylated Fcvariants of the present invention bind an Fc ligand with greateraffinity than the aglycosylated form of the parent Fc polypeptide. SaidFc ligands include but are not limited to FcγRs, C1q, FcRn, and proteinsA and G, and may be from any source including but not limited to human,mouse, rat, rabbit, or monkey, preferably human. In an alternatelypreferred embodiment, the Fc variants are optimized to be more stableand/or more soluble than the aglycosylated form of the parent Fcpolypeptide. An Fc variant that is engineered or predicted to displayany of the aforementioned optimized properties is herein referred to asan “optimized Fc variant”.

The Fc variants of the present invention may be derived from parent Fcpolypeptides that are themselves from a wide range of sources. Theparent Fc polypeptide may be substantially encoded by one or more Fcgenes from any organism, including but not limited to humans, mice,rats, rabbits, camels, llamas, dromedaries, monkeys, preferably mammalsand most preferably humans and mice. In a preferred embodiment, theparent Fc polypeptide composes an antibody, referred to as the parentantibody. The parent antibody may be fully human, obtained for exampleusing transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol8:455-458) or human antibody libraries coupled with selection methods(Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108). The parentantibody need not be naturally occurring. For example, the parentantibody may be an engineered antibody, including but not limited tochimeric antibodies and humanized antibodies (Clark, 2000, Immunol Today21:397-402). The parent antibody may be an engineered variant of anantibody that is substantially encoded by one or more natural antibodygenes. In one embodiment, the parent antibody has been affinity matured,as is known in the art. Alternatively, the antibody has been modified insome other way, for example as described in U.S. Ser. No. 10/339788,filed on Mar. 3, 2003.

The Fc variants of the present invention may be substantially encoded byimmunoglobulin genes belonging to any of the antibody classes. In apreferred embodiment, the Fc variants of the present invention find usein antibodies or Fc fusions that comprise sequences belonging to the IgGclass of antibodies, including IgG1, IgG2, IgG3, or IgG4. In analternate embodiment the Fc variants of the present invention find usein antibodies or Fc fusions that comprise sequences belonging to the IgA(including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes ofantibodies. The Fc variants of the present invention may comprise morethan one protein chain. That is, the present invention may find use inan antibody or Fc fusion that is a monomer or an oligomer, including ahomo- or hetero-oligomer.

In a preferred embodiment, the antibodies of the invention are based onhuman sequences, and thus human sequences are used as the “base”sequences, against which other sequences, such as rat, mouse, and monkeysequences are compared. In order to establish homology to primarysequence or structure, the amino acid sequence of a precursor or parentFc polypeptide is directly compared to the human Fc sequence outlinedherein. After aligning the sequences, using one or more of the homologyalignment programs known in the art (for example using conservedresidues as between species), allowing for necessary insertions anddeletions in order to maintain alignment (i.e., avoiding the eliminationof conserved residues through arbitrary deletion and insertion), theresidues equivalent to particular amino acids in the primary sequence ofhuman Fc are defined. Alignment of conserved residues preferably shouldconserve 100% of such residues. However, alignment of greater than 75%or as little as 50% of conserved residues is also adequate to defineequivalent residues (sometimes referred to as “corresponding residues”).Equivalent residues may also be defined by determining homology at thelevel of tertiary structure for an Fc polypeptide whose tertiarystructure has been determined. Equivalent residues are defined as thosefor which the atomic coordinates of two or more of the main chain atomsof a particular amino acid residue of the parent or precursor (N on N,CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nmafter alignment. Alignment is achieved after the best model has beenoriented and positioned to give the maximum overlap of atomiccoordinates of non-hydrogen protein atoms of the Fc polypeptide.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function or interaction with one or more Fc ligands. Suchcombination may provide additive, synergistic, or novel properties inantibodies or Fc fusions. In one embodiment, the Fc variants of thepresent invention may be combined with other known Fc variants (Duncanet al., 1988, Nature 332:563-564; Lund et al., 1991, J Immunol147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al.,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc NatlAcad Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol Left44:111-117; Lund et al., 1995, Faseb J9:115-119; Jefferis et al., 1996,Immunol Left 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000,J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xuet al., 2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al., 2002, Immunol Left 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490; Hinton et al., 2004, J Biol Chem 279:6213-6216) (U.S.Pat. Nos. 5,624,821; 5,885,573; 6,194,551; PCT WO 00/42072; PCT WO99/58572; 2004/0002587 A1). In an alternate embodiment, the Fc variantsof the present invention are incorporated into an antibody or Fc fusionthat comprises one or more engineered glycoforms. By “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to an Fc polypeptide, wherein said carbohydratecomposition differs chemically from that of a parent Fc polypeptide.Engineered glycoforms may be useful for a variety of purposes, includingbut not limited to enhancing or reducing effector function. Engineeredglycoforms may be generated by a variety of methods known in the art(Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31 140A1;PCT WO 02/30954A1); (Potelligent™ technology [Biowa, Inc., Princeton,N.J.]; GlycoMAb™ glycosylation engineering technology [GLYCARTbiotechnology AG, Zürich, Switzerland]). Many of these techniques arebased on controlling the level of fucosylated and/or bisectingoligosaccharides that are covalently attached to the Fc region, forexample by expressing an Fc polypeptide in various organisms or celllines, engineered or otherwise (for example Lec-13 CHO cells or rathybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [α1,6-fucosyltranserase] and/orβ1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the Fc polypeptide has been expressed. Engineeredglycoform typically refers to the different carbohydrate oroligosaccharide; thus an Fc polypeptide, for example an antibody or Fcfusion, may comprise an engineered glycoform. Alternatively, engineeredglycoform may refer to the Fc polypeptide that comprises the differentcarbohydrate or oligosaccharide. Thus combinations of the Fc variants ofthe present invention with other Fc modifications, as well asundiscovered Fc modifications, are contemplated with the goal ofgenerating novel antibodies or Fc fusions with optimized properties.

The Fc variants of the present invention may find use in an antibody. By“antibody of the present invention” as used herein is meant an antibodythat comprises an Fc variant of the present invention. The presentinvention may, in fact, find use in any protein that comprises Fc, andthus application of the Fc variants of the present invention is notlimited to antibodies. The Fc variants of the present invention may finduse in an Fc fusion. By “Fc fusion of the present invention” as usedherein refers to an Fc fusion that comprises an Fc variant of thepresent invention. Fc fusions may comprise an Fc variant of the presentinvention operably linked to a cytokine, soluble receptor domain,adhesion molecule, ligand, enzyme, peptide, or other protein or proteindomain, and include but are not limited to Fc fusions described in U.S.Pat. Nos. 5,843,725; 6,018,026; 6,291,212; 6,291,646; 6,300,099;6,323,323; PCT WO 00/24782; and in (Chamow et al., 1996, TrendsBiotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200).

Virtually any antigen may be targeted by the antibodies and fusions ofthe present invention, including but not limited to the following listof proteins, subunits, domains, motifs, and epitopes belonging to thefollowing list of proteins: CD2; CD3, CD3E, CD4, CD11, CD11a, CD14,CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33(p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3,IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15,IL-18, IL-23, interferon alpha, interferon beta, interferon gamma;TNF-alpha, TNFbeta2, TNFc, TNFalphabeta, TNF-RI, TNF-RII, FasL, CD27L,CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L,TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin Beta Receptor,TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, integrin beta1,integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin alpha3,integrin alpha4, integrin alpha5, integrin alpha6, integrin alphav,alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4,L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor,B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS(B-lymphocyte Stimulator), complement C5, IgE, factor VII, CD64, CBL,NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4),Tissue Factor, VEGF, VEGFR, endothelin receptor, VLA-4, Hapten NP-cap orNIP-cap, T cell receptor alpha/beta, E-selectin, digoxin, placentalalkaline phosphatase (PLAP) and testicular PLAP-like alkalinephosphatase, transferrin receptor, Carcinoembryonic antigen (CEA),CEACAM5, HMFG PEM, mucin MUC1, MUC18, Heparanase I, human cardiacmyosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associatedantigen CA 125, Prostate specific membrane antigen (PSMA), Highmolecular weight melanoma-associated antigen (HMW-MAA),carcinoma-associated antigen, Gcoprotein IIb/IIIa (GPIIb/IIIa),tumor-associated antigen expressing Lewis Y related carbohydrate, humancytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV,respiratory syncital virus RSV F, RSVF Fgp, VNRintegrin, IL-8,cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIVIIIB gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpessimplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gBenvelope glycoprotein, and Clostridium perfringens toxin.

One skilled in the art will appreciate that the aforementioned list oftargets refers not only to specific proteins and biomolecules, but thebiochemical pathway or pathways that comprise them. For example,reference to CTLA-4 as a target antigen implies that the ligands andreceptors that make up the T cell co-stimulatory pathway, includingCTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands orreceptors that bind these proteins, are also targets. Thus target asused herein refers not only to a specific biomolecule, but the set ofproteins that interact with said target and the members of thebiochemical pathway to which said target belongs. One skilled in the artwill further appreciate that any of the aforementioned target antigens,the ligands or receptors that bind them, or other members of theircorresponding biochemical pathway, may be operably linked to the Fcvariants of the present invention in order to generate an Fc fusion.Thus for example, an Fc fusion that targets EGFR could be constructed byoperably linking an Fc variant to EGF, TGFα, or any other ligand,discovered or undiscovered, that binds EGFR. Accordingly, an Fc variantof the present invention could be operably linked to EGFR in order togenerate an Fc fusion that binds EGF, TGFα, or any other ligand,discovered or undiscovered, that binds EGFR. Thus virtually anypolypeptide, whether a ligand, receptor, or some other protein orprotein domain, including but not limited to the aforementioned targetsand the proteins that compose their corresponding biochemical pathways,may be operably linked to the Fc variants of the present invention todevelop an Fc fusion.

A number of antibodies and Fc fusions that are approved for use, inclinical trials, or in development may benefit from the Fc variants ofthe present invention. Said antibodies and Fc fusions are hereinreferred to as “clinical products and candidates”. Thus in a preferredembodiment, the Fc variants of the present invention may find use in arange of clinical products and candidates. For example, a number ofantibodies that target CD20 may benefit from the Fc variants of thepresent invention. For example the Fc variants of the present inventionmay find use in an antibody that is substantially similar to rituximab(Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat. No.5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-I33 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), and HumaLYM (Intracel). A number of antibodies that targetmembers of the family of epidermal growth factor receptors, includingEGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), maybenefit from the Fc variants of the present invention. For example theFc variants of the present invention may find use in an antibody that issubstantially similar to trastuzumab (Herceptin®, Genentech) (see forexample U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibodyapproved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg™),currently being developed by Genentech; an anti-Her2 antibody describedin U.S. Pat. No. 4,753,894; cetuximab (Erbitux®), Imclone) (U.S. Pat.No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody inclinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No.6,235,883), currently being developed by Abgenix/Immunex/Amgen;HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed byGenmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat.No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60;Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al.,1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research)(PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993,22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993,67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35;Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YMBiosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat.Nos. 5,891,996; 6,506,883; Mateo et al, 1997, Immunotechnology,3(1):71-81); mAb-806 (Ludwig Institue for Cancer Research, MemorialSloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA.100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National CancerInstitute) (PCT WO 0162931A2); and SC100 (Scancell) (PCT WO 01/88138).In another preferred embodiment, the Fc variants of the presentinvention may find use in alemtuzumab (Campath®, Millenium), a humanizedmonoclonal antibody currently approved for treatment of B-cell chroniclymphocytic leukemia. The Fc variants of the present invention may finduse in a variety of antibodies or Fc fusions that are substantiallysimilar to other clinical products and candidates, including but notlimited to muromonab-CD3 (Orthoclone OKT3®)), an anti-CD3 antibodydeveloped by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan(Zevalin®), an anti-CD20 antibody developed by IDEC/Schering AG,gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibodydeveloped by Celltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fcfusion developed by Biogen), abciximab (ReoPro®)), developed byCentocor/Lilly, basiliximab (Simulect®)), developed by Novartis,palivizumab (Synagis®)), developed by MedImmune, infliximab(Remicade®)), an anti-TNFalpha antibody developed by Centocor,adalimumab (Humira®), an anti-TNFalpha antibody developed by Abbott,Humicade™, an anti-TNFalpha antibody developed by Celltech, etanercept(Enbrel®), an anti-TNFalpha Fc fusion developed by Immunex/Amgen,ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, ananti-lL8 antibody being developed by Abgenix, ABX-MA1, an anti-MUC18antibody being developed by Abgenix, Pemtumomab (R1549, ⁹⁰Y-muHMFG1), ananti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1antibody being developed by Antisoma, AngioMab (AS1405), being developedby Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407)being developed by Antisoma, Antegren® (natalizumab), ananti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developedby Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed byBiogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibodybeing developed by Biogen, CAT-152, an anti-TGFβ2 antibody beingdeveloped by Cambridge Antibody Technology, J695, an anti-IL-12 antibodybeing developed by Cambridge Antibody Technology and Abbott, CAT-192, ananti-TGFβ1 antibody being developed by Cambridge Antibody Technology andGenzyme, CAT-213, an anti-Eotaxin1 antibody being developed by CambridgeAntibody Technology, LymphoStat-B™ an anti-Blys antibody being developedby Cambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin™(bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair™ (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva™ (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL 15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide™ (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem™ (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-□5□1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma.

Application of the Fc variants to the aforementioned antibody and Fcfusion clinical products and candidates is not meant to be constrainedto their precise composition. The Fc variants of the present inventionmay be incorporated into the aforementioned clinical candidates andproducts, or into antibodies and Fc fusions that are substantiallysimilar to them. The Fc variants of the present invention may beincorporated into versions of the aforementioned clinical candidates andproducts that are humanized, affinity matured, engineered, or modifiedin some other way. Furthermore, the entire polypeptide of theaforementioned clinical products and candidates need not be used toconstruct a new antibody or Fc fusion that incorporates the Fc variantsof the present invention; for example only the variable region of aclinical product or candidate antibody, a substantially similar variableregion, or a humanized, affinity matured, engineered, or modifiedversion of the variable region may be used. In another embodiment, theFc variants of the present invention may find use in an antibody or Fcfusion that binds to the same epitope, antigen, ligand, or receptor asone of the aforementioned clinical products and candidates.

The Fc variants of the present invention may find use in a wide range ofantibody and Fc fusion products. In one embodiment the antibody or Fcfusion of the present invention is a therapeutic, a diagnostic, or aresearch reagent, preferably a therapeutic. Alternatively, theantibodies and Fc fusions of the present invention may be used foragricultural or industrial uses. In an alternate embodiment, the Fcvariants of the present invention compose a library that may be screenedexperimentally. This library may be a list of nucleic acid or amino acidsequences, or may be a physical composition of nucleic acids orpolypeptides that encode the library sequences. The Fc variant may finduse in an antibody composition that is monoclonal or polyclonal. Theantibodies and Fc fusions of the present invention may be agonists,antagonists, neutralizing, inhibitory, or stimulatory. In a preferredembodiment, the antibodies and Fc fusions of the present invention areused to kill target cells that bear the target antigen, for examplecancer cells. In an alternate embodiment, the antibodies and Fc fusionsof the present invention are used to block, antagonize, or agonize thetarget antigen, for example for antagonizing a cytokine or cytokinereceptor. In an alternately preferred embodiment, the antibodies and Fcfusions of the present invention are used to block, antagonize, oragonize the target antigen and kill the target cells that bear thetarget antigen.

The Fc variants of the present invention may be used for varioustherapeutic purposes. In a preferred embodiment, the Fc variant proteinsare administered to a patient to treat an antibody-related disorder. A“patient” for the purposes of the present invention includes both humansand other animals, preferably mammals and most preferably humans. Thusthe antibodies and Fc fusions of the present invention have both humantherapy and veterinary applications. In the preferred embodiment thepatient is a mammal, and in the most preferred embodiment the patient ishuman. The term “treatment” in the present invention is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor a disease or disorder. Thus, for example, successful administrationof an antibody or Fc fusion prior to onset of the disease results intreatment of the disease. As another example, successful administrationof an optimized antibody or Fc fusion after clinical manifestation ofthe disease to combat the symptoms of the disease comprises treatment ofthe disease. “Treatment” also encompasses administration of an optimizedantibody or Fc fusion protein after the appearance of the disease inorder to eradicate the disease. Successful administration of an agentafter onset and after clinical symptoms have developed, with possibleabatement of clinical symptoms and perhaps amelioration of the disease,comprises treatment of the disease. Those “in need of treatment” includemammals already having the disease or disorder, as well as those proneto having the disease or disorder, including those in which the diseaseor disorder is to be prevented. By “antibody related disorder” or“antibody responsive disorder” or “condition” or “disease” herein aremeant a disorder that may be ameliorated by the administration of apharmaceutical composition comprising an antibody or Fc fusion of thepresent invention. Antibody related disorders include but are notlimited to autoimmune diseases, immunological diseases, infectiousdiseases, inflammatory diseases, neurological diseases, and oncologicaland neoplastic diseases including cancer. By “cancer” and “cancerous”herein refer to or describe the physiological condition in mammals thatis typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to carcinoma, lymphoma, blastoma,sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma,schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophagael cancer, tumors of the biliary tract, as well as head and neckcancer. Furthermore, the Fc variants of the present invention may beused to treat conditions including but not limited to congestive heartfailure (CHF), vasculitis, rosecea, acne, eczema, myocarditis and otherconditions of the myocardium, systemic lupus erythematosus, diabetes,spondylopathies, synovial fibroblasts, and bone marrow stroma; boneloss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer;disuse osteopenia; malnutrition, periodontal disease, Gaucher's disease,Langerhans' cell histiocytosis, spinal cord injury, acute septicarthritis, osteomalacia, Cushing's syndrome, monoostotic fibrousdysplasia, polyostotic fibrous dysplasia, periodontal reconstruction,and bone fractures; sarcoidosis; multiple myeloma; osteolytic bonecancers, breast cancer, lung cancer, kidney cancer and rectal cancer;bone metastasis, bone pain management, and humoral malignanthypercalcemia, ankylosing spondylitisa and other spondyloarthropathies;transplantation rejection, viral infections, hematologic neoplasisas andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticIymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia,tumors of the central nervous system, e.g., brain tumors (glioma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, and tumors of the vascularsystem (angiosarcoma and hemagiopericytoma), osteoporosis, hepatitis,HIV, AIDS, spondyloarthritis, rheumatoid arthritis, inflammatory boweldiseases (IBD), sepsis and septic shock, Crohn's Disease, psoriasis,schleraderma, graft versus host disease (GVHD), allogenic islet graftrejection, hematologic malignancies, such as multiple myeloma (MM),myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML),inflammation associated with tumors, peripheral nerve injury ordemyelinating diseases.

In one embodiment, an antibody or Fc fusion of the present invention isadministered to a patient having a disease involving inappropriateexpression of a protein. Within the scope of the present invention thisis meant to include diseases and disorders characterized by aberrantproteins, due for example to alterations in the amount of a proteinpresent, the presence of a mutant protein, or both. An overabundance maybe due to any cause, including but not limited to overexpression at themolecular level, prolonged or accumulated appearance at the site ofaction, or increased activity of a protein relative to normal. Includedwithin this definition are diseases and disorders characterized by areduction of a protein. This reduction may be due to any cause,including but not limited to reduced expression at the molecular level,shortened or reduced appearance at the site of action, mutant forms of aprotein, or decreased activity of a protein relative to normal. Such anoverabundance or reduction of a protein can be measured relative tonormal expression, appearance, or activity of a protein, and saidmeasurement may play an important role in the development and/orclinical testing of the antibodies and Fc fusions of the presentinvention.

In one embodiment, an antibody or Fc fusion of the present invention isthe only therapeutically active agent administered to a patient.Alternatively, the antibody or Fc fusion of the present invention isadministered in combination with one or more other therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, or othertherapeutic agents. Such molecules are suitably present in combinationin amounts that are effective for the purpose intended. The skilledmedical practitioner can determine empirically the appropriate dose ordoses of other therapeutic agents useful herein. The antibodies and Fcfusions of the present invention may be administered concomitantly withone or more other therapeutic regimens. For example, an antibody or Fcfusion of the present invention may be administered to the patient alongwith chemotherapy, radiation therapy, or both chemotherapy and radiationtherapy. In one embodiment, the antibody or Fc fusion of the presentinvention may be administered in conjunction with one or more antibodiesor Fc fusions, which may or may not comprise an Fc variant of thepresent invention.

In one embodiment, the antibodies and Fc fusions of the presentinvention are administered with a chemotherapeutic agent. By“chemotherapeutic agent” as used herein is meant a chemical compounduseful in the treatment of cancer. Examples of chemotherapeutic agentsinclude but are not limited to alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, poffiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; thymidylate synthase inhibitor (such as Tomudex); cox-2inhibitors, such as celicoxib (CELEBREX®) or MK-0966 (VIOXX®); andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such as antiestrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY 117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; and Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardtet al., (ed.): 247-267, Humana Press, 1985. The prodrugs that may finduse with the present invention include but are not limited tophosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use withthe antibodies and Fc fusions of the present invention include but arenot limited to any of the aforementioned chemotherapeutic agents.

The antibodies and Fc fusions of the present invention may be combinedwith other therapeutic regimens. For example, in one embodiment, thepatient to be treated with the antibody or Fc fusion may also receiveradiation therapy. Radiation therapy can be administered according toprotocols commonly employed in the art and known to the skilled artisan.Such therapy includes but is not limited to cesium, iridium, iodine, orcobalt radiation. The radiation therapy may be whole body irradiation,or may be directed locally to a specific site or tissue in or on thebody, such as the lung, bladder, or prostate. Typically, radiationtherapy is administered in pulses over a period of time from about 1 to2 weeks. The radiation therapy may, however, be administered over longerperiods of time. For instance, radiation therapy may be administered topatients having head and neck cancer for about 6 to about 7 weeks.Optionally, the radiation therapy may be administered as a single doseor as multiple, sequential doses. The skilled medical practitioner candetermine empirically the appropriate dose or doses of radiation therapyuseful herein. In accordance with another embodiment of the invention,the antibody or Fc fusion of the present invention and one or more otheranti-cancer therapies are employed to treat cancer cells ex vivo. It iscontemplated that such ex vivo treatment may be useful in bone marrowtransplantation and particularly, autologous bone marrowtransplantation. For instance, treatment of cells or tissue(s)containing cancer cells with antibody or Fc fusion and one or more otheranti-cancer therapies, such as described above, can be employed todeplete or substantially deplete the cancer cells prior totransplantation in a recipient patient. It is of course contemplatedthat the antibodies and Fc fusions of the invention can be employed incombination with still other therapeutic techniques such as surgery.

In an alternate embodiment, the antibodies and Fc fusions of the presentinvention are administered with a cytokine. By “cytokine” as used hereinis meant a generic term for proteins released by one cell populationthat act on another cell as intercellular mediators. Examples of suchcytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture, and biologically active equivalents of the native sequencecytokines.

A variety of other therapeutic agents may find use for administrationwith the antibodies and Fc fusions of the present invention. In oneembodiment, the antibody or Fc fusion is administered with ananti-angiogenic agent. By “anti-angiogenic agent” as used herein ismeant a compound that blocks, or interferes to some degree, thedevelopment of blood vessels. The anti-angiogenic factor may, forinstance, be a small molecule or a protein, for example an antibody, Fcfusion, or cytokine, that binds to a growth factor or growth factorreceptor involved in promoting angiogenesis. The preferredanti-angiogenic factor herein is an antibody that binds to VascularEndothelial Growth Factor (VEGF). In an alternate embodiment, theantibody or Fc fusion is administered with a therapeutic agent thatinduces or enhances adaptive immune response, for example an antibodythat targets CTLA-4. In an alternate embodiment, the antibody or Fcfusion is administered with a tyrosine kinase inhibitor. By “tyrosinekinase inhibitor” as used herein is meant a molecule that inhibits tosome extent tyrosine kinase activity of a tyrosine kinase. Examples ofsuch inhibitors include but are not limited to quinazolines, such as PD153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines;pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3-d)pyrimidines; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophenemoieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g. thosethat bind to ErbB-encoding nucleic acid); quinoxalines (U.S. Pat. No.5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering A G); pan-ErbB inhibitors such asC1-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate(ST1571,Gleevec®; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen);ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11(Imclone); or as described in any of the following patent publications:U.S. Pat. No. 5,804,396; PCT WO 99/09016 (American Cyanimid); PCT WO98/43960 (American Cyanamid); PCT WO 97/38983 (Warner-Lambert); PCT WO99/06378 (Warner-Lambert); PCT WO 99/06396 (Warner-Lambert); PCT WO96/30347 (Pfizer, Inc); PCT WO 96/33978 (AstraZeneca); PCT WO96/3397(AstraZeneca); PCT WO 96/33980 (AstraZeneca), gefitinib (IRESSA™,ZD1839, AstraZeneca), and OSI-774 (Tarceva™, OSIPharmaceuticals/Genentech).

A variety of linkers may find use in the present invention to generateFc fusions (see definition above) or antibody- or Fc fusion- conjugates(see definition below). By “linker”, “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof, herein is meanta molecule or group of molecules (such as a monomer or polymer) thatconnects two molecules and often serves to place the two molecules in apreferred configuration. A number of strategies may be used tocovalently link molecules together. These include, but are not limitedto polypeptide linkages between N- and C-termini of proteins or proteindomains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. Choosing a suitable linker for a specific case where twopolypeptide chains are to be connected depends on various parameters,including but not limited to the nature of the two polypeptide chains(e.g., whether they naturally oligomerize), the distance between the N-and the C-termini to be connected if known, and/or the stability of thelinker towards proteolysis and oxidation. Furthermore, the linker maycontain amino acid residues that provide flexibility. Thus, the linkerpeptide may predominantly include the following amino acid residues:Gly, Ser, Ala, or Thr. The linker peptide should have a length that isadequate to link two molecules in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity. Suitable lengths for this purpose include at least oneand not more than 30 amino acid residues. Preferably, the linker is fromabout 1 to 30 amino acids in length, with linkers of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19 and 20 amino acids inlength being preferred. In addition, the amino acid residues selectedfor inclusion in the linker peptide should exhibit properties that donot interfere significantly with the activity of the polypeptide. Thus,the linker peptide on the whole should not exhibit a charge that wouldbe inconsistent with the activity of the polypeptide, or interfere withinternal folding, or form bonds or other interactions with amino acidresidues in one or more of the monomers that would seriously impede thebinding of receptor monomer domains. Useful linkers includeglycine-serine polymers (including, for example, (GS)n, (GSGGS)n(GGGGS)n and (GGGS)n, where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers such as the tether for the shaker potassium channel, and a largevariety of other flexible linkers, as will be appreciated by those inthe art. Glycine-serine polymers are preferred since both of these aminoacids are relatively unstructured, and therefore may be able to serve asa neutral tether between components. Secondly, serine is hydrophilic andtherefore able to solubilize what could be a globular glycine chain.Third, similar chains have been shown to be effective in joiningsubunits of recombinant proteins such as single chain antibodies.Suitable linkers may also be identified by screening databases of knownthree-dimensional structures for naturally occurring motifs that canbridge the gap between two polypeptide chains. In a preferredembodiment, the linker is not immunogenic when administered in a humanpatient. Thus linkers may be chosen such that they have lowimmunogenicity or are thought to have low immunogenicity. For example, alinker may be chosen that exists naturally in a human. In a preferredembodiment the linker has the sequence of the hinge region of anantibody, that is the sequence that links the antibody Fab and Fcregions; alternatively the linker has a sequence that comprises part ofthe hinge region, or a sequence that is substantially similar to thehinge region of an antibody. Another way of obtaining a suitable linkeris by optimizing a simple linker, e.g., (Gly4Ser)n, through randommutagenesis. Alternatively, once a suitable polypeptide linker isdefined, additional linker polypeptides can be created to select aminoacids that more optimally interact with the domains being linked. Othertypes of linkers that may be used in the present invention includeartificial polypeptide linkers and inteins. In another embodiment,disulfide bonds are designed to link the two molecules. In anotherembodiment, linkers are chemical cross-linking agents. For example, avariety of bifunctional protein coupling agents may be used, includingbut not limited to N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1971, Science 238:1098.Chemical linkers may enable chelation of an isotope. For example,Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyidiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see PCT WO 94/11026).The linker may be cleavable, facilitating release of the cytotoxic drugin the cell. For example, an acid-labile linker, peptidase-sensitivelinker, dimethyl linker or disulfide-containing linker (Chari et al.,1992, Cancer Research 52: 127-131) may be used. Alternatively, a varietyof nonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use to link the Fc variants of the present invention toa fusion partner to generate an Fc fusion, or to link the antibodies andFc fusions of the present invention to a conjugate.

In one embodiment, the antibody or Fc fusion of the present invention isconjugated or operably linked to another therapeutic compound, referredto herein as a conjugate. The conjugate may be a cytotoxic agent, achemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosinekinase inhibitor, a toxin, a radioisotope, or other therapeuticallyactive agent. Chemotherapeutic agents, cytokines, anti-angiogenicagents, tyrosine kinase inhibitors, and other therapeutic agents havebeen described above, and all of these aforemention therapeutic agentsmay find use as antibody or Fc fusion conjugates. In an alternateembodiment, the antibody or Fc fusion is conjugated or operably linkedto a toxin, including but not limited to small molecule toxins andenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Small moleculetoxins include but are not limited to calicheamicin, maytansine (U.S.Pat. No. 5,208,020), trichothene, and CC1065. In one embodiment of theinvention, the antibody or Fc fusion is conjugated to one or moremaytansine molecules (e.g. about 1 to about 10 maytansine molecules perantibody molecule). Maytansine may, for example, be converted toMay-SS-Me which may be reduced to May-SH3 and reacted with modifiedantibody or Fc fusion (Chari et al., 1992, Cancer Research 52: 127-131)to generate a maytansinoid-antibody or maytansinoid-Fc fusion conjugate.Another conjugate of interest comprises an antibody or Fc fusionconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structural analogues ofcalicheamicin that may be used include but are not limited to γ₁ ¹, α₂¹, α₃, N-acetyl-γ₁ ¹, PSAG, and Θ¹ ₁, (Hinman et al., 1993, CancerResearch 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928)(U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; 5,773,001). Dolastatin10 analogs such as auristatin E (AE) and monomethylauristatin E (MMAE)may find use as conjugates for the Fc variants of the present invention(Doronina et al., 2003, Nat Biotechnol 21 (7):778-84; Francisco et al.,2003 Blood 102(4):1458-65). Useful enyzmatically active toxins includebut are not limited to diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes. See, for example, PCT WO93/21232. The present invention further contemplates a conjugate orfusion formed between an antibody or Fc fusion of the present inventionand a compound with nucleolytic activity, for example a ribonuclease orDNA endonuclease such as a deoxyribonuclease (DNase).

In an alternate embodiment, an antibody or Fc fusion of the presentinvention may be conjugated or operably linked to a radioisotope to forma radioconjugate. A variety of radioactive isotopes are available forthe production of radioconjugate antibodies and Fc fusions. Examplesinclude, but are not limited to, At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², and radioactive isotopes of Lu.

In yet another embodiment, an antibody or Fc fusion of the presentinvention may be conjugated to a “receptor” (such streptavidin) forutilization in tumor pretargeting wherein the antibody-receptor or Fcfusion-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide). In an alternateembodiment, the antibody or Fc fusion is conjugated or operably linkedto an enzyme in order to employ Antibody Dependent Enzyme MediatedProdrug Therapy (ADEPT). ADEPT may be used by conjugating or operablylinking the antibody or Fc fusion to a prodrug-activating enzyme thatconverts a prodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO81/01145) to an active anti-cancer drug. See, for example, PCT WO88/07378 and U.S. Pat. No. 4,975,278. The enzyme component of theimmunoconjugate useful for ADEPT includes any enzyme capable of actingon a prodrug in such a way so as to covert it into its more active,cytotoxic form. Enzymes that are useful in the method of this inventioninclude but are not limited to alkaline phosphatase useful forconverting phosphate-containing prodrugs into free drugs; arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic 5-fluorocytosine intothe anti-cancer drug, 5-fluorouracil; proteases, such as serratiaprotease, thermolysin, subtilisin, carboxypeptidases and cathepsins(such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as .beta.-galactosidase andneuramimidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized with.alpha.-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458).Antibody-abzyme and Fc fusion-abzyme conjugates can be prepared fordelivery of the abzyme to a tumor cell population.

Other modifications of the antibodies and Fc fusions of the presentinvention are contemplated herein. For example, the antibody or Fcfusion may be linked to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,or copolymers of polyethylene glycol and polypropylene glycol.

Pharmaceutical compositions are contemplated wherein an antibody or Fcfusion of the present invention and and one or more therapeuticallyactive agents are formulated. Formulations of the antibodies and Fcfusions of the present invention are prepared for storage by mixing saidantibody or Fc fusion having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients orstabilizers.(Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed.,1980), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, acetate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, corn and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). In a preferred embodiment, the pharmaceuticalcomposition that comprises the antibody or Fc fusion of the presentinvention is in a water-soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferrably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

The antibodies and Fc fusions disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the antibody orFc fusion are prepared by methods known in the art, such as described inEpstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al.,1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. Nos. 4,485,045;4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. The components of the liposomeare commonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Particularly useful liposomes canbe generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent or other therapeuticallyactive agent is optionally contained within the liposome (Gabizon etal., 1989, J National Cancer Inst 81:1484).

The antibodies, Fc fusions, and other therapeutically active agents mayalso be entrapped in microcapsules prepared by methods including but notlimited to coacervation techniques, interfacial polymerization (forexample using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (forexample, liposomes, albumin microspheres, microemulsions, nano-particlesand nanocapsules), and macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymer, which matrices are in the form of shaped articles,e.g. films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease® (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-glycolide(PLG).

The concentration of the therapeutically active antibody or Fc fusion ofthe present invention in the formulation may vary from about 0.1 to 100weight %. In a preferred embodiment, the concentration of the antibodyor Fc fusion is in the range of 0.003 to 1.0 molar. In order to treat apatient, a therapeutically effective dose of the antibody or Fc fusionof the present invention may be administered. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.01 to 100 mg/kg of bodyweight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight,with 1 to 10 mg/kg being preferred. As is known in the art, adjustmentsfor antibody or Fc fusion degradation, systemic versus localizeddelivery, and rate of new protease synthesis, as well as the age, bodyweight, general health, sex, diet, time of administration, druginteraction and the severity of the condition may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

Administration of the pharmaceutical composition comprising an antibodyor Fc fusion of the present invention, preferably in the form of asterile aqueous solution, may be done in a variety of ways, including,but not limited to orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (e.g., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary (e.g.,AERx® inhalable technology commercially available from Aradigm, orInhance™ pulmonary delivery system commercially available from InhaleTherapeutics), vaginally, parenterally, rectally, or intraocularly. Insome instances, for example for the treatment of wounds, inflammation,etc., the antibody or Fc fusion may be directly applied as a solution orspray. As is known in the art, the pharmaceutical composition may beformulated accordingly depending upon the manner of introduction.

Engineering Methods

The present invention provides engineering methods that may be used togenerate Fc variants. A principal obstacle that has hindered previousattempts at Fc engineering is that only random attempts at modificationhave been possible, due in part to the inefficiency of engineeringstrategies and methods, and to the low-throughput nature of antibodyproduction and screening. The present invention describes engineeringmethods that overcome these shortcomings. A variety of designstrategies, computational screening methods, library generation methods,and experimental production and screening methods are contemplated.These strategies, approaches, techniques, and methods may be appliedindividually or in various combinations to engineer optimized Fcvariants.

Design Strategies

The most efficient approach to generating Fc variants that are optimizedfor a desired property is to direct the engineering efforts toward thatgoal. Accordingly, the present invention teaches design strategies thatmay be used to engineer optimized Fc variants. The use of a designstrategy is meant to guide Fc engineering, but is not meant to constrainan Fc variant to a particular optimized property based on the designstrategy that was used to engineer it. At first thought this may seemcounterintuitive; however its validity is derived from the enormouscomplexity of subtle interactions that determine the structure,stability, solubility, and function of proteins and protein-proteincomplexes. Although efforts can be made to predict which proteinpositions, residues, interactions, etc. are important for a design goal,often times critical ones are not predictable. Effects on proteinstructure, stability, solubility, and function, whether favorable orunfavorable, are often unforeseen. Yet there are innumerable amino acidmodifications that are detrimental or deleterious to proteins. Thusoften times the best approach to engineering comes from generation ofprotein variants that are focused generally towards a design goal but donot cause detrimental effects. In this way, a principal objective of adesign strategy may be the generation of quality diversity. At asimplistic level this can be thought of as stacking the odds in one'sfavor. As an example, perturbation of the Fc carbohydrate or aparticular domain-domain angle, as described below, are valid designstrategies for generating optimized Fc variants, despite the fact thathow carbohydrate and domain-domain angles determine the properties of Fcis not well understood. By reducing the number of detrimental amino acidmodifications that are screened, i.e. by screening quality diversity,these design strategies become practical. Thus the true value of thedesign strategies taught in the present invention is their ability todirect engineering efforts towards the generation of valuable Fcvariants. The specific value of any one resulting variant is determinedafter experimentation.

One design strategy for engineering Fc variants is provided in whichinteraction of Fc with some Fc ligand is altered by engineering aminoacid modifications at the interface between Fc and said Fc ligand. Fcligands herein may include but are not limited to FcγRs, C1q, FcRn,protein A or G, and the like. By exploring energetically favorablesubstitutions at Fc positions that impact the binding interface,variants can be engineered that sample new interface conformations, someof which may improve binding to the Fc ligand, some of which may reduceFc ligand binding, and some of which may have other favorableproperties. Such new interface conformations could be the result of, forexample, direct interaction with Fc ligand residues that form theinterface, or indirect effects caused by the amino acid modificationssuch as perturbation of side chain or backbone conformations. Variablepositions may be chosen as any positions that are believed to play animportant role in determining the conformation of the interface. Forexample, variable positions may be chosen as the set of residues thatare within a certain distance, for example 5 Angstroms (Å), preferrablybetween 1 and 10 Å, of any residue that makes direct contact with the Fcligand.

An additional design strategy for generating Fc variants is provided inwhich the conformation of the Fc carbohydrate at N297 is optimized.Optimization as used in this context is meant to includes conformationaland compositional changes in the N297 carbohydrate that result in adesired property, for example increased or reduced affinity for an FcγR.Such a strategy is supported by the observation that the carbohydratestructure and conformation dramatically affect Fc/FcγR and Fc/C1qbinding (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol Chem276:45539-45547.; Radaev et al., 2001, J Biol Chem 276:16478-16483;Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al.,2003, J Biol Chem 278:3466-3473). However the carbohydrate makes nospecific contacts with FcγRs. By exploring energetically favorablesubstitutions at positions that interact with carbohydrate, a qualitydiversity of variants can be engineered that sample new carbohydrateconformations, some of which may improve and some of which may reducebinding to one or more Fc ligands. While the majority of mutations nearthe Fc/carbohydrate interface appear to alter carbohydrate conformation,some mutations have been shown to alter the glycosylation composition(Lund et al., 1996, J Immunol 157:4963-4969; Jefferis et al., 2002,Immunol Lett 82:57-65).

Another design strategy for generating Fc variants is provided in whichthe angle between the Cγ2 and Cγ3 domains is optimized Optimization asused in this context is meant to describe conformational changes in theCγ2-Cγ3 domain angle that result in a desired property, for exampleincreased or reduced affinity for an FcγR. This angle is an importantdeterminant of Fc/FcγR affinity (Radaev et al., 2001, J Biol Chem276:16478-16483), and a number of mutations distal to the Fc/FcγRinterface affect binding potentially by modulating it (Shields et al.,2001, J Biol Chem 276:6591-6604). By exploring energetically favorablesubstitutions positions that appear to play a key role in determiningthe Cγ2-Cγ3 angle and the flexibility of the domains relative to oneanother, a quality diversity of variants can be designed that sample newangles and levels of flexibility, some of which may be optimized for adesired Fc property.

Another design strategy for generating Fc variants is provided in whichFc is reengineered to eliminate the structural and functional dependenceon glycosylation. This design strategy involves the optimization of Fcstructure, stability, solubility, and/or Fc function (for exampleaffinity of Fc for one or more Fc ligands) in the absence of the N297carbohydrate. In one approach, positions that are exposed to solvent inthe absence of glycosylation are engineered such that they are stable,structurally consistent with Fc structure, and have no tendency toaggregate. The Cγ2 is the only unpaired Ig domain in the antibody (seeFIG. 1). Thus the N297 carbohydrate covers up the exposed hydrophobicpatch that would normally be the interface for a protein-proteininteraction with another Ig domain, maintaining the stability andstructural integrity of Fc and keeping the Cγ2 domains from aggregatingacross the central axis. Approaches for optimizing aglycosylated Fc mayinvolve but are not limited to designing amino acid modifications thatenhance aglycoslated Fc stability and/or solubility by incorporatingpolar and/or charged residues that face inward towards the Cγ2-Cγ2 dimeraxis, and by designing amino acid modifications that directly enhancethe aglycosylated Fc/FcγR interface or the interface of aglycosylated Fcwith some other Fc ligand.

An additional design strategy for engineering Fc variants is provided inwhich the conformation of the Cγ2 domain is optimized Optimization asused in this context is meant to describe conformational changes in theCγ2 domain angle that result in a desired property, for exampleincreased or reduced affinity for an FcγR. By exploring energeticallyfavorable substitutions at Cγ2 positions that impact the Cγ2conformation, a quality diversity of variants can be engineered thatsample new Cγ2 conformations, some of which may achieve the design goal.Such new Cγ2 conformations could be the result of, for example,alternate backbone conformations that are sampled by the variant.Variable positions may be chosen as any positions that are believed toplay an important role in determining Cγ2 structure, stability,solubility, flexibility, function, and the like. For example, Cγ2hydrophobic core residues, that is Cγ2 residues that are partially orfully sequestered from solvent, may be reengineered. Alternatively,noncore residues may be considered, or residues that are deemedimportant for determining backbone structure, stability, or flexibility.

An additional design strategy for Fc optimization is provided in whichbinding to an FcγR, complement, or some other Fc ligand is altered bymodifications that modulate the electrostatic interaction between Fc andsaid Fc ligand. Such modifications may be thought of as optimization ofthe global electrostatic character of Fc, and include replacement ofneutral amino acids with a charged amino acid, replacement of a chargedamino acid with a neutral amino acid, or replacement of a charged aminoacid with an amino acid of opposite charge (i.e. charge reversal). Suchmodifications may be used to effect changes in binding affinity betweenan Fc and one or more Fc ligands, for example FcγRs. In a preferredembodiment, positions at which electrostatic substitutions might affectbinding are selected using one of a variety of well known methods forcalculation of electrostatic potentials. In the simplest embodiment,Coulomb's law is used to generate electrostatic potentials as a functionof the position in the protein. Additional embodiments include the useof Debye-Huckel scaling to account for ionic strength effects, and moresophisticated embodiments such as Poisson-Boltzmann calculations. Suchelectrostatic calculations may highlight positions and suggest specificamino acid modifications to achieve the design goal. In some cases,these substitutions may be anticipated to variably affect binding todifferent Fc ligands, for example to enhance binding to activating FcγRswhile decreasing binding affinity to inhibitory FcγRs.

Computational Screening

A principal obstacle to obtaining valuable Fc variants is the difficultyin predicting what amino acid modifications, out of the enormous numberof possibilities, will achieve the desired goals. Indeed one of theprinciple reasons that previous attempts at Fc engineering have failedto produce Fc variants of significant clinical value is that approachesto Fc engineering have thus far involved hit-or-miss approaches. Thepresent invention provides computational screening methods that enablequantitative and systematic engineering of Fc variants. These methodstypically use atomic level scoring functions, side chain rotamersampling, and advanced optimization methods to accurately capture therelationships between protein sequence, structure, and function.Computational screening enables exploration of the entire sequence spaceof possibilities at target positions by filtering the enormous diversitywhich results. Variant libraries that are screened computationally areeffectively enriched for stable, properly folded, and functionalsequences, allowing active optimization of Fc for a desired goal.Because of the overlapping sequence constraints on protein structure,stability, solubility, and function, a large number of the candidates ina library occupy “wasted” sequence space. For example, a large fractionof sequence space encodes unfolded, misfolded, incompletely folded,partially folded, or aggregated proteins. This is particularly relevantfor Fc engineering because Ig domains are small beta sheet structures,the engineering of which has proven extremely demanding (Quinn et al.,1994, Proc Natl Acad Sci USA 91:8747-8751; Richardson et al., 2002, ProcNatl Acad Sci USA 99:2754-2759). Even seemingly harmless substitutionson the surface of a beta sheet can cause severe packing conflicts,dramatically disrupting folding equilibrium (Smith et al., 1995, Science270:980-982); incidentally, alanine is one of the worst beta sheetformers (Minor et al., 1994, Nature 371:264-267). The determinants ofbeta sheet stability and specificity are a delicate balance between anextremely large number of subtle interactions. Computational screeningenables the generation of libraries that are composed primarily ofproductive sequence space, and as a result increases the chances ofidentifying proteins that are optimized for the design goal. In effect,computational screening yields an increased hit-rate, thereby decreasingthe number of variants that must be screened experimentally. Anadditional obstacle to Fc engineering is the need for active design ofcorrelated or coupled mutations. For example, the greatest Fc/FcγRaffinity enhancement observed thus far is S298A/E333A/K334A, obtained bycombining three better binders obtained separately in an alanine scan(Shields et al., 2001, J Biol Chem 276:6591-6604). Computationalscreening is capable of generating such a three-fold variant in oneexperiment instead of three separate ones, and furthermore is able totest the functionality of all 20 amino acids at those positions insteadof just alanine. Computational screening deals with such complexity byreducing the combinatorial problem to an experimentally tractable size.

Computational screening, viewed broadly, has four steps: 1) selectionand preparation of the protein template structure or structures, 2)selection of variable positions, amino acids to be considered at thosepositions, and/or selection of rotamers to model considered amino acids,3) energy calculation, and 4) combinatorial optimization. In moredetail, the process of computational screening can be described asfollows. A three-dimensional structure of a protein is used as thestarting point. The positions to be optimized are identified, which maybe the entire protein sequence or subset(s) thereof. Amino acids thatwill be considered at each position are selected. In a preferredembodiment, each considered amino acid may be represented by a discreteset of allowed conformations, called rotamers. Interaction energies arecalculated between each considered amino acid and each other consideredamino acid, and the rest of the protein, including the protein backboneand invariable residues. In a preferred embodiment, interaction energiesare calculated between each considered amino acid side chain rotamer andeach other considered amino acid side chain rotamer and the rest of theprotein, including the protein backbone and invariable residues. One ormore combinatorial search algorithms are then used to identify thelowest energy sequence and/or low energy sequences.

In a preferred embodiment, the computational screening method used issubstantially similar to Protein Design Automation® (PDA®) technology,as is described in U.S. Pat. Nos. 6,188,965; 6,269,312; 6,403,312; U.S.Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No. 10/218,102;PCT WO 98/07254; PCT WO 01/40091; and PCT WO 02/25588. In anotherpreferred embodiment, a computational screening method substantiallysimilar to Sequence Prediction Algorithm™ (SPA™) technology is used, asis described in (Raha et al., 2000, Protein Sci 9:1106-1119), U.S. Ser.No. 09/877,695, and U.S. Ser. No. 10/071,859. In another preferredembodiment, the computational screening methods described in U.S. Ser.No. 10/339788, filed on Mar. 3, 2003, entitled “ANTIBODY OPTIMIZATION”,are used. In some embodiments, combinations of different computationalscreening methods are used, including combinations of PDA® technologyand SPA™ technology, as well as combinations of these computationalmethods in combination with other design tools. Similarly, thesecomputational methods can be used simultaneously or sequentially, in anyorder.

A template structure is used as input into the computational screeningcalculations. By “template structure” herein is meant the structuralcoordinates of part or all of a protein to be optimized. The templatestructure may be any protein for which a three dimensional structure(that is, three dimensional coordinates for a set of the protein'satoms) is known or may be calculated, estimated, modeled, generated, ordetermined. The three dimensional structures of proteins may bedetermined using methods including but not limited to X-raycrystallographic techniques, nuclear magnetic resonance (NMR)techniques, de novo modeling, and homology modeling. If optimization isdesired for a protein for which the structure has not been solvedexperimentally, a suitable structural model may be generated that mayserve as the template for computational screening calculations. Methodsfor generating homology models of proteins are known in the art, andthese methods find use in the present invention. See for example, Luo,et al. 2002, Protein Sci 11: 1218-1226, Lehmann & Wyss, 2001, Curr OpinBiotechnol 12(4):371-5.; Lehmann et. al., 2000, Biochim Biophys Acta1543(2):408-415; Rath & Davidson, 2000, Protein Sci, 9(12):2457-69;Lehmann et al., 2000 Protein Eng 13(1):49-57; Desjarlais & Berg, 1993,Proc Natl Acad Sci USA 90(6):2256-60; Desjarlais & Berg, 1992, Proteins12(2):101-4; Henikoff & Henikoff, 2000, Adv Protein Chem 54:73-97;Henikoff & Henikoff, 1994, J Mol Biol 243(4):574-8; Morea et al., 2000,Methods 20:267-269. Protein/protein complexes may also be obtained usingdocking methods. Suitable protein structures that may serve as templatestructures include, but are not limited to, all of those found in theProtein Data Base compiled and serviced by the Research Collaboratoryfor Structural Bioinformatics (RCSB, formerly the Brookhaven NationalLab).

The template structure may be of a protein that occurs naturally or isengineered. The template structure may be of a protein that issubstantially encoded by a protein from any organism, with human, mouse,rat, rabbit, and monkey preferred. The template structure may compriseany of a number of protein structural forms. In a preferred embodimentthe template structure comprises an Fc region or a domain or fragment ofFc. In an alternately preferred embodiment the template structurecomprises Fc or a domain or fragment of Fc bound to one or more Fcligands, with an Fc/FcγR complex being preferred. The Fc in the templatestructure may be glycosylated or unglycosylated. The template structuremay comprise more than one protein chain. The template structure mayadditionally contain nonprotein components, including but not limited tosmall molecules, substrates, cofactors, metals, water molecules,prosthetic groups, polymers and carbohydrates. In a preferredembodiment, the template structure is a plurality or set of templateproteins, for example an ensemble of structures such as those obtainedfrom NMR. Alternatively, the set of template structures is generatedfrom a set of related proteins or structures, or artificially createdensembles. The composition and source of the template structure dependson the engineering goal. For example, for enhancement of human Fc/FcγRaffinity, a human Fc/FcγR complex structure or derivative thereof may beused as the template structure. Alternatively, the uncomplexed Fcstructure may be used as the template structure. If the goal is toenhance affinity of a human Fc for a mouse FcγR, the template structuremay be a structure or model of a human Fc bound to a mouse FcγR.

The template structure may be modified or altered prior to designcalculations. A variety of methods for template structure preparationare described in U.S. Pat. Nos. 6,188,965; 6,269,312; 6,403,312; U.S.Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No. 09/877,695;U.S. Ser. No. 10/071,859, U.S. Ser. No. 10/218,102; PCT WO 98/07254; PCTWO 01/40091; and PCT WO 02/25588. For example, in a preferredembodiment, explicit hydrogens may be added if not included within thestructure. In an alternate embodiment, energy minimization of thestructure is run to relax strain, including strain due to van der Waalsclashes, unfavorable bond angles, and unfavorable bond lengths.Alternatively, the template structure is altered using other methods,such as manually, including directed or random perturbations. It is alsopossible to modify the template structure during later steps ofcomputational screening, including during the energy calculation andcombinatorial optimization steps. In an alternate embodiment, thetemplate structure is not modified before or during computationalscreening calculations.

Once a template structure has been obtained, variable positions arechosen. By “variable position” herein is meant a position at which theamino acid identity is allowed to be altered in a computationalscreening calculation. As is known in the art, allowing amino acidmodifications to be considered only at certain variable positionsreduces the complexity of a calculation and enables computationalscreening to be more directly tailored for the design goal. One or moreresidues may be variable positions in computational screeningcalculations. Positions that are chosen as variable positions may bethose that contribute to or are hypothesized to contribute to theprotein property to be optimized, for example Fc affinity for an FcγR,Fc stability, Fc solubility, and so forth. Residues at variablepositions may contribute favorably or unfavorably to a specific proteinproperty. For example, a residue at an Fc/FcγR interface may be involvedin mediating binding, and thus this position may be varied in designcalculations aimed at improving Fc/FcγR affinity. As another example, aresidue that has an exposed hydrophobic side chain may be responsiblefor causing unfavorable aggregation, and thus this position may bevaried in design calculations aimed at improving solubility. Variablepositions may be those positions that are directly involved ininteractions that are determinants of a particular protein property. Forexample, the FcγR binding site of Fc may be defined to include allresidues that contact that particular FγcR. By “contact” herein is meantsome chemical interaction between at least one atom of an Fc residuewith at least one atom of the bound FcγR, with chemical interactionincluding, but not limited to van der Waals interactions, hydrogen bondinteractions, electrostatic interactions, and hydrophobic interactions.In an alternative embodiment, variable positions may include thosepositions that are indirectly involved in a protein property, i.e. suchpositions may be proximal to residues that are known to or hypothesizedto contribute to an Fc property. For example, the FcγR binding site ofan Fc may be defined to include all Fc residues within a certaindistance, for example 4-10 Å, of any Fc residue that is in van der Waalscontact with the FcγR. Thus variable positions in this case may bechosen not only as residues that directly contact the FcγR, but alsothose that contact residues that contact the FcγR and thus influencebinding indirectly. The specific positions chosen are dependent on thedesign strategy being employed.

One or more positions in the template structure that are not variablemay be floated. By “floated position” herein is meant a position atwhich the amino acid conformation but not the amino acid identity isallowed to vary in a computational screening calculation. In oneembodiment, the floated position may have the parent amino acididentity. For example, floated positions may be positions that arewithin a small distance, for example 5 Å, of a variable positionresidue. In an alternate embodiment, a floated position may have anon-parent amino acid identity. Such an embodiment may find use in thepresent invention, for example, when the goal is to evaluate theenergetic or structural outcome of a specific mutation.

Positions that are not variable or floated are fixed. By “fixedPosition” herein is meant a position at which the amino acid identityand the conformation are held constant in a computational screeningcalculation. Positions that may be fixed include residues that are notknown to be or hypothesized to be involved in the property to beoptimized. In this case the assumption is that there is little ornothing to be gained by varying these positions. Positions that arefixed may also include positions whose residues are known orhypothesized to be important for maintaining proper folding, structure,stability, solubility, and/or biological function. For example,positions may be fixed for residues that interact with a particular Fcligand or residues that encode a glycosylation site in order to ensurethat binding to the Fc ligand and proper glycosylation respectively arenot perturbed. Likewise, if stability is being optimized, it may bebeneficial to fix positions that directly or indirectly interact with anFc ligand, for example an FcγR, so that binding is not perturbed. Fixedpositions may also include structurally important residues such ascysteines participating in disulfide bridges, residues critical fordetermining backbone conformation such as proline or glycine, criticalhydrogen bonding residues, and residues that form favorable packinginteractions.

The next step in computational screening is to select a set of possibleamino acid identities that will be considered at each particularvariable position. This set of possible amino acids is herein referredto as “considered amino acids” at a variable position. “Amino acids” asused herein refers to the set of natural 20 amino acids and anynonnatural or synthetic analogues. In one embodiment, all 20 naturalamino acids are considered. Alternatively, a subset of amino acids, oreven only one amino acid is considered at a given variable position. Aswill be appreciated by those skilled in the art, there is acomputational benefit to considering only certain amino acid identitiesat variable positions, as it decreases the combinatorial complexity ofthe search. Furthermore, considering only certain amino acids atvariable positions may be used to tailor calculations toward specificdesign strategies. For example, for solubility optimization ofaglycosylated Fc, it may be beneficial to allow only polar amino acidsto be considered at nonpolar Fc residues that are exposed to solvent inthe absence of carbohydrate. Nonnatural amino acids, including syntheticamino acids and analogues of natural amino acids, may also be consideredamino acids. For example see Chin et al., 2003, Science,301(5635):964-7; and Chin et al., 2003, Chem Biol.10(6):511-9.

A wide variety of methods may be used, alone or in combination, toselect which amino acids will be considered at each position. Forexample, the set of considered amino acids at a given variable positionmay be chosen based on the degree of exposure to solvent. Hydrophobic ornonpolar amino acids typically reside in the interior or core of aprotein, which are inaccessible or nearly inaccessible to solvent. Thusat variable core positions it may be beneficial to consider only ormostly nonpolar amino acids such as alanine, valine, isoleucine,leucine, phenylalanine, tyrosine, tryptophan, and methionine.Hydrophilic or polar amino acids typically reside on the exterior orsurface of proteins, which have a significant degree of solventaccessibility. Thus at variable surface positions it may be beneficialto consider only or mostly polar amino acids such as alanine, serine,threonine, aspartic acid, asparagine, glutamine, glutamic acid,arginine, lysine and histidine. Some positions are partly exposed andpartly buried, and are not clearly protein core or surface positions, ina sense serving as boundary residues between core and surface residues.Thus at such variable boundary positions it may be beneficial toconsider both nonpolar and polar amino acids such as alanine, serine,threonine, aspartic acid, asparagine, glutamine, glutamic acid,arginine, lysine histidine, valine, isoleucine, leucine, phenylalanine,tyrosine, tryptophan, and methionine. Determination of the degree ofsolvent exposure at variable positions may be by subjective evaluationor visual inspection of the template structure by one skilled in the artof protein structural biology, or by using a variety of algorithms thatare known in the art. Selection of amino acid types to be considered atvariable positions may be aided or determined wholly by computationalmethods, such as calculation of solvent accessible surface area, orusing algorithms that assess the orientation of the Cα-Cβ vectorsrelative to a solvent accessible surface, as outlined in U.S. Pat. Nos.6,188,965; 6,269,312; 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No.09/927,790; U.S. Ser. No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091;and PCT WO 02/25588. In one embodiment, each variable position may beclassified explicitly as a core, surface, or boundary position or aclassification substantially similar to core, surface, or boundary.

In an alternate embodiment, selection of the set of amino acids allowedat variable positions may be hypothesis-driven. Hypotheses for whichamino acid types should be considered at variable positions may bederived by a subjective evaluation or visual inspection of the templatestructure by one skilled in the art of protein structural biology. Forexample, if it is suspected that a hydrogen bonding interaction may befavorable at a variable position, polar residues that have the capacityto form hydrogen bonds may be considered, even if the position is in thecore. Likewise, if it is suspected that a hydrophobic packinginteraction may be favorable at a variable position, nonpolar residuesthat have the capacity to form favorable packing interactions may beconsidered, even if the position is on the surface. Other examples ofhypothesis-driven approaches may involve issues of backbone flexibilityor protein fold. As is known in the art, certain residues, for exampleproline, glycine, and cysteine, play important roles in proteinstructure and stability. Glycine enables greater backbone flexibilitythan all other amino acids, proline constrains the backbone more thanall other amino acids, and cysteines may form disulfide bonds. It maytherefore be beneficial to include one or more of these amino acid typesto achieve a desired design goal. Alternatively, it may be beneficial toexclude one or more of these amino acid types from the list ofconsidered amino acids.

In an alternate embodiment, subsets of amino acids may be chosen tomaximize coverage. In this case, additional amino acids with propertiessimilar to that in the template structure may be considered at variablepositions. For example, if the residue at a variable position in thetemplate structure is a large hydrophobic residue, additional largehydrophobic amino acids may be considered at that position.Alternatively, subsets of amino acids may be chosen to maximizediversity. In this case, amino acids with properties dissimilar to thosein the template structure may be considered at variable positions. Forexample, if the residue at a variable position in the template is alarge hydrophobic residue, amino acids that are small, polar, etc. maybe considered.

As is known in the art, some computational screening methods requireonly the identity of considered amino acids to be determined duringdesign calculations. That is, no information is required concerning theconformations or possible conformations of the amino acid side chains.Other preferred methods utilize a set of discrete side chainconformations, called rotamers, which are considered for each aminoacid. Thus, a set of rotamers may be considered at each variable andfloated position. Rotamers may be obtained from published rotamerlibraries (see for example, Lovel et al., 2000, Proteins: StructureFunction and Genetics 40:389-408; Dunbrack & Cohen, 1997, ProteinScience 6:1661-1681; DeMaeyer et al., 1997, Folding and Design 2:53-66;Tuffery et al., 1991, J Biomol Struct Dyn 8:1267-1289, Ponder &Richards, 1987, J Mol Biol 193:775-791). As is known in the art, rotamerlibraries may be backbone-independent or backbone-dependent. Rotamersmay also be obtained from molecular mechanics or ab initio calculations,and using other methods. In a preferred embodiment, a flexible rotamermodel is used (see Mendes et al., 1999, Proteins: Structure, Function,and Genetics 37:530-543). Similarly, artificially generated rotamers maybe used, or augment the set chosen for each amino acid and/or variableposition. In one embodiment, at least one conformation that is not lowin energy is included in the list of rotamers. In an alternateembodiment, the rotamer of the variable position residue in the templatestructure is included in the list of rotamers allowed for that variableposition. In an alternate embodiment, only the identity of each aminoacid considered at variable positions is provided, and no specificconformational states of each amino acid are used during designcalculations. That is, use of rotamers is not essential forcomputational screening.

Experimental information may be used to guide the choice of variablepositions and/or the choice of considered amino acids at variablepositions. As is known in the art, mutagenesis experiments are oftencarried out to determine the role of certain residues in proteinstructure and function, for example, which protein residues play a rolein determining stability, or which residues make up the interface of aprotein-protein interaction. Data obtained from such experiments areuseful in the present invention. For example, variable positions forFc/FcγR affinity enhancement could involve varying all positions atwhich mutation has been shown to affect binding. Similarly, the resultsfrom such an experiment may be used to guide the choice of allowed aminoacid types at variable positions. For example, if certain types of aminoacid substitutions are found to be favorable, similar types of thoseamino acids may be considered. In one embodiment, additional amino acidswith properties similar to those that were found to be favorableexperimentally may be considered at variable positions. For example, ifexperimental mutation of a variable position at an Fc/FcγR interface toa large hydrophobic residue was found to be favorable, the user maychoose to include additional large hydrophobic amino acids at thatposition in the computational screen. As is known in the art, displayand other selection technologies may be coupled with random mutagenesisto generate a list or lists of amino acid substitutions that arefavorable for the selected property. Such a list or lists obtained fromsuch experimental work find use in the present invention. For example,positions that are found to be invariable in such an experiment may beexcluded as variable positions in computational screening calculations,whereas positions that are found to be more acceptable to mutation orrespond favorably to mutation may be chosen as variable positions.Similarly, the results from such experiments may be used to guide thechoice of allowed amino acid types at variable positions. For example,if certain types of amino acids arise more frequently in an experimentalselection, similar types of those amino acids may be considered. In oneembodiment, additional amino acids with properties similar to those thatwere found to be favorable experimentally may be considered at variablepositions. For example, if selected mutations at a variable positionthat resides at an Fc/FcγR interface are found to be uncharged polaramino acids, the user may choose to include additional uncharged polaramino acids, or perhaps charged polar amino acids, at that position.

Sequence information may also be used to guide choice of variablepositions and/or the choice of amino acids considered at variablepositions. As is known in the art, some proteins share a commonstructural scaffold and are homologous in sequence. This information maybe used to gain insight into particular positions in the protein family.As is known in the art, sequence alignments are often carried out todetermine which protein residues are conserved and which are notconserved. That is to say, by comparing and contrasting alignments ofprotein sequences, the degree of variability at a position may beobserved, and the types of amino acids that occur naturally at positionsmay be observed. Data obtained from such analyses are useful in thepresent invention. The benefit of using sequence information to choosevariable positions and considered amino acids at variable positions areseveral fold. For choice of variable positions, the primary advantage ofusing sequence information is that insight may be gained into whichpositions are more tolerant and which are less tolerant to mutation.Thus sequence information may aid in ensuring that quality diversity,i.e. mutations that are not deleterious to protein structure, stability,etc., is sampled computationally. The same advantage applies to use ofsequence information to select amino acid types considered at variablepositions. That is, the set of amino acids that occur in a proteinsequence alignment may be thought of as being pre-screened by evolutionto have a higher chance than random for being compatible with aprotein's structure, stability, solubility, function, etc. Thus higherquality diversity is sampled computationally. A second benefit of usingsequence information to select amino acid types considered at variablepositions is that certain alignments may represent sequences that may beless immunogenic than random sequences. For example, if the amino acidsconsidered at a given variable position are the set of amino acids whichoccur at that position in an alignment of human protein sequences, thoseamino acids may be thought of as being pre-screened by nature forgenerating no or low immune response if the optimized protein is used asa human therapeutic.

The source of the sequences may vary widely, and include one or more ofthe known databases, including but not limited to the Kabat database(Johnson & Wu, 2001, Nucleic Acids Res 29:205-206; Johnson & Wu, 2000,Nucleic Acids Res 28:214-218), the IMGT database (IMGT, theinternational ImMunoGeneTics information system®; Lefranc et al., 1999,Nucleic Acids Res 27:209-212; Ruiz et al., 2000 Nucleic Acids Re.28:219-221; Lefranc et al., 2001, Nucleic Acids Res 29:207-209; Lefrancet al., 2003, Nucleic Acids Res 31:307-310), and VBASE, SwissProt,GenBank and Entrez, and EMBL Nucleotide Sequence Database. Proteinsequence information can be obtained, compiled, and/or generated fromsequence alignments of naturally occurring proteins from any organism,including but not limited to mammals. Protein sequence information canbe obtained from a database that is compiled privately. There arenumerous sequence-based alignment programs and methods known in the art,and all of these find use in the present invention for generation ofsequence alignments of proteins that comprise Fc and Fc ligands.

Once alignments are made, sequence information can be used to guidechoice of variable positions. Such sequence information can relate thevariability, natural or otherwise, of a given position. Variabilityherein should be distinguished from variable position. Variabilityrefers to the degree to which a given position in a sequence alignmentshows variation in the types of amino acids that occur there. Variableposition, to reiterate, is a position chosen by the user to vary inamino acid identity during a computational screening calculation.Variability may be determined qualitatively by one skilled in the art ofbioinformatics. There are also methods known in the art toquantitatively determine variability that may find use in the presentinvention. The most preferred embodiment measures Information Entropy orShannon Entropy. Variable positions can be chosen based on sequenceinformation obtained from closely related protein sequences, orsequences that are less closely related.

The use of sequence information to choose variable positions finds broaduse in the present invention. For example, if an Fc/FcγR interfaceposition in the template structure is tryptophan, and tryptophan isobserved at that position in greater than 90% of the sequences in analignment, it may be beneficial to leave that position fixed. Incontrast, if another interface position is found to have a greater levelof variability, for example if five different amino acids are observedat that position with frequencies of approximately 20% each, thatposition may be chosen as a variable position. In another embodiment,visual inspection of aligned protein sequences may substitute for or aidvisual inspection of a protein structure. Sequence information can alsobe used to guide the choice of amino acids considered at variablepositions. Such sequence information can relate to how frequently anamino acid, amino acids, or amino acid types (for example polar ornonpolar, charged or uncharged) occur, naturally or otherwise, at agiven position. In one embodiment, the set of amino acids considered ata variable position may comprise the set of amino acids that is observedat that position in the alignment. Thus, the position-specific alignmentinformation is used directly to generate the list of considered aminoacids at a variable position in a computational screening calculation.Such a strategy is well known in the art; see for example Lehmann &Wyss, 2001, Curr Opin Biotechnol 12(4):371-5; Lehmann et al., 2000,Biochim Biophys Acta 1543(2):408-415; Rath & Davidson, 2000, ProteinSci, 9(12):2457-69; Lehmann et al., 2000, Protein Eng 13(1):49-57;Desjarlais & Berg, 1993, Proc Natl Acad Sci USA 90(6):2256-60;Desjarlais & Berg, 1992, Proteins 12(2):101-4; Henikoff & Henikoff,2000, Adv Protein Chem 54:73-97; Henikoff & Henikoff, 1994, J Mol Biol243(4):574-8. In an alternate embodiment, the set of amino acidsconsidered at a variable position or positions may comprise a set ofamino acids that is observed most frequently in the alignment. Thus, acertain criteria is applied to determine whether the frequency of anamino acid or amino acid type warrants its inclusion in the set of aminoacids that are considered at a variable position. As is known in theart, sequence alignments may be analyzed using statistical methods tocalculate the sequence diversity at any position in the alignment andthe occurrence frequency or probability of each amino acid at aposition. Such data may then be used to determine which amino acidstypes to consider. In the simplest embodiment, these occurrencefrequencies are calculated by counting the number of times an amino acidis observed at an alignment position, then dividing by the total numberof sequences in the alignment. In other embodiments, the contribution ofeach sequence, position or amino acid to the counting procedure isweighted by a variety of possible mechanisms. In a preferred embodiment,the contribution of each aligned sequence to the frequency statistics isweighted according to its diversity weighting relative to othersequences in the alignment. A common strategy for accomplishing this isthe sequence weighting system recommended by Henikoff and Henikoff(Henikoff & Henikoff, 2000, Adv Protein Chem 54:73-97; Henikoff &Henikoff, 1994, J Mol Biol 243:574-8. In a preferred embodiment, thecontribution of each sequence to the statistics is dependent on itsextent of similarity to the target sequence, i.e. the template structureused, such that sequences with higher similarity to the target sequenceare weighted more highly. Examples of similarity measures include, butare not limited to, sequence identity, BLOSUM similarity score, PAMmatrix similarity score, and BLAST score. In an alternate embodiment,the contribution of each sequence to the statistics is dependent on itsknown physical or functional properties. These properties include, butare not limited to, thermal and chemical stability, contribution toactivity, and solubility. For example, when optimizing aglycosylated Fcfor solubility, those sequences in an alignment that are known to bemost soluble (for example see Ewert et al., 2003, J Mol Biol325:531-553), will contribute more heavily to the calculatedfrequencies.

Regardless of what criteria are applied for choosing the set of aminoacids in a sequence alignment to be considered at variable positions,use of sequence information to choose considered amino acids finds broaduse in the present invention. For example, to optimize Fc solubility byreplacing exposed nonpolar surface residues, considered amino acids maybe chosen as the set of amino acids, or a subset of those amino acidswhich meet some criteria, that are observed at that position in analignment of protein sequences. As another example, one or more aminoacids may be added or subtracted subjectively from a list of amino acidsderived from a sequence alignment in order to maximize coverage. Forexample, additional amino acids with properties similar to those thatare found in a sequence alignment may be considered at variablepositions. For example, if an Fc position that is known to orhypothesized to bind an FcγR is observed to have uncharged polar aminoacids in a sequence alignment, the user may choose to include additionaluncharged polar amino acids in a computational screening calculation, orperhaps charged polar amino acids, at that position.

In one embodiment, sequence alignment information is combined withenergy calculation, as discussed below. For example, pseudo energies canbe derived from sequence information to generate a scoring function. Theuse of a sequence-based scoring function may assist in significantlyreducing the complexity of a calculation. However, as is appreciated bythose skilled in the art, the use of a sequence-based scoring functionalone may be inadequate because sequence information can often indicatemisleading correlations between mutations that may in reality bestructurally conflicting. Thus, in a preferred embodiment, astructure-based method of energy calculation is used, either alone or incombination with a sequence-based scoring function. That is, preferredembodiments do not rely on sequence alignment information alone as theanalysis step.

Energy calculation refers to the process by which amino acidmodifications are scored. The energies of interaction are measured byone or more scoring functions. A variety of scoring functions find usein the present invention for calculating energies. Scoring functions mayinclude any number of potentials, herein referred to as the energy termsof a scoring function, including but not limited to a van der Waalspotential, a hydrogen bond potential, an atomic solvation potential orother solvation models, a secondary structure propensity potential, anelectrostatic potential, a torsional potential, and an entropypotential. At least one energy term is used to score each variable orfloated position, although the energy terms may differ depending on theposition, considered amino acids, and other considerations. In oneembodiment, a scoring function using one energy term is used. In themost preferred embodiment, energies are calculated using a scoringfunction that contains more than one energy term, for example describingvan der Waals, solvation, electrostatic, and hydrogen bond interactions,and combinations thereof. In additional embodiments, additional energyterms include but are not limited to entropic terms, torsional energies,and knowledge-based energies.

A variety of scoring functions are described in U.S. Pat. Nos.6,188,965; 6,269,312; 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No.09/927,790; U.S. Ser. No. 09/877,695; U.S. Ser. No. 10/071,859, U.S.Ser. No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO02/25588. As will be appreciated by those skilled in the art, scoringfunctions need not be limited to physico-chemical energy terms. Forexample, knowledge-based potentials may find use in the computationalscreening methodology of the present invention. Such knowledge-basedpotentials may be derived from protein sequence and/or structurestatistics including but not limited to threading potentials, referenceenergies, pseudo energies, homology-based energies, and sequence biasesderived from sequence alignments. In a preferred embodiment, a scoringfunction is modified to include models for immunogenicity, such asfunctions derived from data on binding of peptides to MHC (MajorHtocompatability Complex), that may be used to identify potentiallyimmunogenic sequences (see for example U.S. Ser. No. 09/903,378; U.S.Ser. No. 10/039,170; U.S. Ser. No. 60/222,697; U.S. Ser. No. 10/339788;PCT WO 01/21823; and PCT WO 02/00165). In one embodiment, sequencealignment information can be used to score amino acid substitutions. Forexample, comparison of protein sequences, regardless of whether thesource of said proteins is human, monkey, mouse, or otherwise, may beused to suggest or score amino acid mutations in the computationalscreening methodology of the present invention. In one embodiment, as isknown in the art, one or more scoring functions may be optimized or“trained” during the computational analysis, and then the analysisre-run using the optimized system. Such altered scoring functions may beobtained for example, by training a scoring function using experimentaldata. As will be appreciated by those skilled in the art, a number offorce fields, which are comprised of one or more energy terms, may serveas scoring functions. Force fields include but are not limited to abinitio or quantum mechanical force fields, semi-empirical force fields,and molecular mechanics force fields. Scoring functions that areknowledge-based or that use statistical methods may find use in thepresent invention. These methods may be used to assess the match betweena sequence and a three-dimensional protein structure, and hence may beused to score amino acid substitutions for fidelity to the proteinstructure. In one embodiment, molecular dynamics calculations may beused to computationally screen sequences by individually calculatingmutant sequence scores.

There are a variety of ways to represent amino acids in order to enableefficient energy calculation. In a preferred embodiment, consideredamino acids are represented as rotamers, as described previously, andthe energy (or score) of interaction of each possible rotamer at eachvariable and floated position with the other variable and floatedrotamers, with fixed position residues, and with the backbone structureand any non-protein atoms, is calculated. In a preferred embodiment, twosets of interaction energies are calculated for each side chain rotamerat every variable and floated position: the interaction energy betweenthe rotamer and the fixed atoms (the “singles” energy), and theinteraction energy between the variable and floated positions rotamerand all other possible rotamers at every other variable and floatedposition (the “doubles” energy). In an alternate embodiment, singles anddoubles energies are calculated for fixed positions as well as forvariable and floated positions. In an alternate embodiment, consideredamino acids are not represented as rotamers.

An important component of computational screening is the identificationof one or.more sequences that have a favorable score, i.e. are low inenergy. Determining a set of low energy sequences from an extremelylarge number of possibilities is nontrivial, and to solve this problem acombinatorial optimization algorithm is employed. The need for acombinatorial optimization algorithm is illustrated by examining thenumber of possibilities that are considered in a typical computationalscreening calculation. The discrete nature of rotamer sets allows asimple calculation of the number of possible rotameric sequences for agiven design problem. A backbone of length n with m possible rotamersper position will have m^(n) possible rotamer sequences, a number thatgrows exponentially with sequence length. For very simple calculations,it is possible to examine each possible sequence in order to identifythe optimal sequence and/or one or more favorable sequences. However,for a typical design problem, the number of possible sequences (up to10⁸⁰ or more) is sufficiently large that examination of each possiblesequence is intractable. A variety of combinatorial optimizationalgorithms may then be used to identify the optimum sequence and/or oneor more favorable sequences. Combinatorial optimization algorithms maybe divided into two classes: (1) those that are guaranteed to return theglobal minimum energy configuration if they converge, and (2) those thatare not guaranteed to return the global minimum energy configuration,but which will always return a solution. Examples of the first class ofalgorithms include but are not limited to Dead-End Elimination (DEE) andBranch & Bound (B&B) (including Branch and Terminate) (Gordon & Mayo,1999, Structure Fold Des 7:1089-98). Examples of the second class ofalgorithms include, but are not limited to, Monte Carlo (MC),self-consistent mean field (SCMF), Boltzmann sampling (Metropolis etal., 1953, J Chem Phys 21:1087), simulated annealing (Kirkpatrick etal., 1983, Science, 220:671-680), genetic algorithm (GA), and Fast andAccurate Side-Chain Topology and Energy Refinement (FASTER) (Desmet, etal., 2002, Proteins, 48:31-43). A combinatorial optimization algorithmmay be used alone or in conjunction with another combinatorialoptimization algorithm.

In one embodiment of the present invention, the strategy for applying acombinatorial optimization algorithm is to find the global minimumenergy configuration. In an alternate embodiment, the strategy is tofind one or more low energy or favorable sequences. In an alternateembodiment, the strategy is to find the global minimum energyconfiguration and then find one or more low energy or favorablesequences. For example, as outlined in U.S. Ser. No. 6,269,312,preferred embodiments utilize a Dead End Elimination(DEE) step and aMonte Carlo step. In other embodiments, tabu search algorithms are usedor combined with DEE and/or Monte Carlo, among other search methods (seeModern Heuristic Search Methods, edited by V. J. Rayward-Smith et al.,1996, John Wiley & Sons Ltd.; U.S. Ser. No. 10/218,102; and PCT WO02/25588). In another preferred embodiment, a genetic algorithm may beused; see for example U.S. Ser. No. 09/877,695 and U.S. Ser. No.10/071,859. As another example, as is more fully described in U.S. Pat.Nos. 6,188,965; 6,269,312; 6,403,312; U.S. Ser. No. 09/782,004; U.S.Ser. No. 09/927,790; U.S. Ser. No. 10/218,102; PCT WO 98/07254; PCT WO01/40091; and PCT WO 02/25588, the global optimum may be reached, andthen further computational processing may occur, which generatesadditional optimized sequences. In the simplest embodiment, designcalculations are not combinatorial. That is, energy calculations areused to evaluate amino acid substitutions individually at singlevariable positions. For other calculations it is preferred to evaluateamino acid substitutions at more than one variable position. In apreferred embodiment, all possible interaction energies are calculatedprior to combinatorial optimization. In an alternatively preferredembodiment, energies may be calculated as needed during combinatorialoptimization.

Library Generation

The present invention provides methods for generating libraries that maysubsequently be screened experimentally to single out optimized Fcvariants. By “library” as used herein is meant a set of one or more Fcvariants. Library may refer to the set of variants in any form. In oneembodiment, the library is a list of nucleic acid or amino acidsequences, or a list of nucleic acid or amino acid substitutions atvariable positions. For example, the examples used to illustrate thepresent invention below provide libraries as amino acid substitutions atvariable positions. In one embodiment, a library is a list of at leastone sequence that are Fc variants optimized for a desired property. Forexample see, Filikov et al., 2002, Protein Sci 11:1452-1461 and Luo etal., 2002, Protein Sci 11:1218-1226. In an alternate embodiment, alibrary may be defined as a combinatorial list, meaning that a list ofamino acid substitutions is generated for each variable position, withthe implication that each substitution is to be combined with all otherdesigned substitutions at all other variable positions. In this case,expansion of the combination of all possibilities at all variablepositions results in a large explicitly defined library. A library mayrefer to a physical composition of polypeptides that comprise the Fcregion or some domain or fragment of the Fc region. Thus a library mayrefer to a physical composition of antibodies or Fc fusions, either inpurified or unpurified form. A library may refer to a physicalcomposition of nucleic acids that encode the library sequences. Saidnucleic acids may be the genes encoding the library members, the genesencoding the library members with any operably linked nucleic acids, orexpression vectors encoding the library members together with any otheroperably linked regulatory sequences, selectable markers, fusionconstructs, and/or other elements. For example, the library may be a setof mammalian expression vectors that encode Fc library members, theprotein products of which may be subsequently expressed, purified, andscreened experimentally. As another example, the library may be adisplay library. Such a library could, for example, comprise a set ofexpression vectors that encode library members operably linked to somefusion partner that enables phage display, ribosome display, yeastdisplay, bacterial surface display, and the like.

The library may be generated using the output sequence or sequences fromcomputational screening. As discussed above, computationally generatedlibraries are significantly enriched in stable, properly folded, andfunctional sequences relative to randomly generated libraries. As aresult, computational screening increases the chances of identifyingproteins that are optimized for the design goal. The set of sequences ina library is generally, but not always, significantly different from theparent sequence, although in some cases the library preferably containsthe parent sequence. As is known in the art, there are a variety of waysthat a library may be derived from the output of computational screeningcalculations. For example, methods of library generation described inU.S. Pat. No. 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No.09/927,790; U.S. Ser. No. 10/218,102; PCT WO 01/40091; and PCT WO02/25588 find use in the present invention. In one embodiment, sequencesscoring within a certain range of the global optimum sequence may beincluded in the library. For example, all sequences within 10 kcal/molof the lowest energy sequence could be used as the library. In analternate embodiment, sequences scoring within a certain range of one ormore local minima sequences may be used. In a preferred embodiment, thelibrary sequences are obtained from a filtered set. Such a list or setmay be generated by a variety of methods, as is known in the art, forexample using an algorithm such as Monte Carlo, B&B, or SCMF. Forexample, the top 10³ or the top 10⁵ sequences in the filtered set maycomprise the library. Alternatively, the total number of sequencesdefined by the combination of all mutations may be used as a cutoffcriterion for the library. Preferred values for the total number ofrecombined sequences range from 10 to 10²⁰, particularly preferredvalues range from 100 to 10⁹. Alternatively, a cutoff may be enforcedwhen a predetermined number of mutations per position is reached. Insome embodiments, sequences that do not make the cutoff are included inthe library. This may be desirable in some situations, for instance toevaluate the approach to library generation, to provide controls orcomparisons, or to sample additional sequence space. For example, theparent sequence may be included in the library, even if it does not makethe cutoff.

Clustering algorithms may be useful for classifying sequences derived bycomputational screening methods into representative groups. For example,the methods of clustering and their application described in U.S. Ser.No. 10/218,102 and PCT WO 02/25588, find use in the present invention.Representative groups may be defined, for example, by similarity.Measures of similarity include, but are not limited to sequencesimilarity and energetic similarity. Thus the output sequences fromcomputational screening may be clustered around local minima, referredto herein as clustered sets of sequences. For example, sets of sequencesthat are close in sequence space may be distinguished from other sets.In one embodiment, coverage within one or a subset of clustered sets maybe maximized by including in the library some, most, or all of thesequences that make up one or more clustered sets of sequences. Forexample, it may be advantageous to maximize coverage within the one,two, or three lowest energy clustered sets by including the majority ofsequences within these sets in the library. In an alternate embodiment,diversity across clustered sets of sequences may be sampled by includingwithin a library only a subset of sequences within each clustered set.For example, all or most of the clustered sets could be broadly sampledby including the lowest energy sequence from each clustered set in thelibrary.

Sequence information may be used to guide or filter computationallyscreening results for generation of a library. As discussed, bycomparing and contrasting alignments of protein sequences, the degree ofvariability at a position and the types of amino acids which occurnaturally at that position may be observed. Data obtained from suchanalyses are useful in the present invention. The benefits of usingsequence information have been discussed, and those benefits applyequally to use of sequence information to guide library generation. Theset of amino acids that occur in a sequence alignment may be thought ofas being pre-screened by evolution to have a higher chance than randomat being compatible with a protein's structure, stability, solubility,function, and immunogenicity. The variety of sequence sources, as wellas the methods for generating sequence alignments that have beendiscussed, find use in the application of sequence information toguiding library generation. Likewise, as discussed above, variouscriteria may be applied to determine the importance or weight of certainresidues in an alignment. These methods also find use in the applicationof sequence information to guide library generation. Using sequenceinformation to guide library generation from the results ofcomputational screening finds broad use in the present invention. In oneembodiment, sequence information is used to filter sequences fromcomputational screening output. That is to say, some substitutions aresubtracted from the computational output to generate the library. Forexample the resulting output of a computational screening calculation orcalculations may be filtered so that the library includes only thoseamino acids, or a subset of those amino acids that meet some criteria,for example that are observed at that position in an alignment ofsequences. In an alternate embodiment, sequence information is used toadd sequences to the computational screening output. That is to say,sequence information is used to guide the choice of additional aminoacids that are added to the computational output to generate thelibrary. For example, the output set of amino acids for a given positionfrom a computational screening calculation may be augmented to includeone or more amino acids that are observed at that position in analignment of protein sequences. In an alternate embodiment, based onsequence alignment information, one or more amino acids may be added toor subtracted from the computational screening sequence output in orderto maximize coverage or diversity. For example, additional amino acidswith properties similar to those that are found in a sequence alignmentmay be added to the library. For example, if a position is observed tohave uncharged polar amino acids in a sequence alignment, additionaluncharged polar amino acids may be included in the library at thatposition.

Libraries may be processed further to generate subsequent libraries. Inthis way, the output from a computational screening calculation orcalculations may be thought of as a primary library. This primarylibrary may be combined with other primary libraries from othercalculations or other libraries, processed using subsequentcalculations, sequence information, or other analyses, or processedexperimentally to generate a subsequent library, herein referred to as asecondary library. As will be appreciated from this description, the useof sequence information to guide or filter libraries, discussed above,is itself one method of generating secondary libraries from primarylibraries. Generation of secondary libraries gives the user greatercontrol of the parameters within a library. This enables more efficientexperimental screening, and may allow feedback from experimental resultsto be interpreted more easily, providing a more efficientdesign/experimentation cycle.

There are a wide variety of methods to generate secondary libraries fromprimary libraries. For example, U.S. Ser. No. 10/218,102 and PCT WO02/25588, describes methods for secondary library generation that finduse in the present invention. Typically some selection step occurs inwhich a primary library is processed in some way. For example, in oneembodiment a selection step occurs wherein some set of primary sequencesare chosen to form the secondary library. In an alternate embodiment, aselection step is a computational step, again generally including aselection step, wherein some subset of the primary library is chosen andthen subjected to further computational analysis, including both furthercomputational screening as well as techniques such as “in silico”shuffling or recombination (see, for example U.S. Pat. Nos. 5,830,721;5,811,238; 5,605,793; and 5,837,458, error-prone PCR, for example usingmodified nucleotides; known mutagenesis techniques including the use ofmulti-cassettes; and DNA shuffling (Crameri et al., 1998, Nature391:288-291; Coco et al., 2001, Nat Biotechnol 19:354-9; Coco et al.,2002, Nat Biotechnol, 20:1246-50), heterogeneous DNA samples (U.S. Pat.No. 5,939,250); ITCHY (Ostermeier et al., 1999, Nat Biotechnol17:1205-1209); StEP (Zhao et al., 1998, Nat Biotechnol 16:258-261), GSSM(U.S. Pat. Nos. 6,171,820 and 5,965,408); in vivo homologousrecombination, ligase assisted gene assembly, end-complementary PCR,profusion (Roberts & Szostak, 1997, Proc Natl Acad Sci USA94:12297-12302); yeast/bacteria surface display (Lu et al., 1995,Biotechnology 13:366-372); Seed & Aruffo, 1987, Proc Natl Acad Sci USA84(10):3365-3369; Boder & Wittrup, 1997, Nat Biotechnol 15:553-557). Inan alternate embodiment, a selection step occurs that is an experimentalstep, for example any of the library screening steps below, wherein somesubset of the primary library is chosen and then recombinedexperimentally, for example using one of the directed evolution methodsdiscussed below, to form a secondary library. In a preferred embodiment,the primary library is generated and processed as outlined in U.S. Pat.No. 6,403,312.

Generation of secondary and subsequent libraries finds broad use in thepresent invention. In one embodiment, different primary libraries may becombined to generate a secondary or subsequent library. In anotherembodiment, secondary libraries may be generated by sampling sequencediversity at highly mutatable or highly conserved positions. The primarylibrary may be analyzed to determine which amino acid positions in thetemplate protein have high mutational frequency, and which positionshave low mutational frequency. For example, positions in a protein thatshow a great deal of mutational diversity in computational screening maybe fixed in a subsequent round of design calculations. A filtered set ofthe same size as the first would now show diversity at positions thatwere largely conserved in the first library. Alternatively, thesecondary library may be generated by varying the amino acids at thepositions that have high numbers of mutations, while keeping constantthe positions that do not have mutations above a certain frequency.

This discussion is not meant to constrain generation of librariessubsequent to primary libraries to secondary libraries. As will beappreciated, primary and secondary libraries may be processed further togenerate tertiary libraries, quaternary libraries, and so on. In thisway, library generation is an iterative process. For example, tertiarylibraries may be constructed using a variety of additional steps appliedto one or more secondary libraries; for example, further computationalprocessing may occur, secondary libraries may be recombined, or subsetsof different secondary libraries may be combined. In a preferredembodiment, a tertiary library may be generated by combining secondarylibraries. For example, primary and/or secondary libraries that analyzeddifferent parts of a protein may be combined to generate a tertiarylibrary that treats the combined parts of the protein. In an alternateembodiment, the variants from a primary library may be combined with thevariants from another primary library to provide a combined tertiarylibrary at lower computational cost than creating a very long filteredset. These combinations may be used, for example, to analyze largeproteins, especially large multi-domain proteins, of which Fc is anexample. Thus the above description of secondary library generationapplies to generating any library subsequent to a primary library, theend result being a final library that may screened experimentally toobtain protein variants optimized for a design goal. These examples arenot meant to constrain generation of secondary libraries to anyparticular application or theory of operation for the present invention.Rather, these examples are meant to illustrate that generation ofsecondary libraries, and subsequent libraries such as tertiary librariesand so on, is broadly useful in computational screening methodology forlibrary generation.

Experimental Production and Screening

The present invention provides methods for producing and screeninglibraries of Fc variants. The described methods are not meant toconstrain the present invention to any particular application or theoryof operation. Rather, the provided methods are meant to illustrategenerally that one or more Fc variants or one or more libraries of Fcvariants may be produced and screened experimentally to obtain optimizedFc variants. Fc variants may be produced and screened in any context,whether as an Fc region as precisely defined herein, a domain orfragment thereof, or a larger polypeptide that comprises Fc such as anantibody or Fc fusion. General methods for antibody molecular biology,expression, purification, and screening are described in AntibodyEngineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689;Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: ALaboratory Manual by Harlow & Lane, New York: Cold Spring HarborLaboratory Press, 1988.

In one embodiment of the present invention, the library sequences areused to create nucleic acids that encode the member sequences, and thatmay then be cloned into host cells, expressed and assayed, if desired.Thus, nucleic acids, and particularly DNA, may be made that encode eachmember protein sequence. These practices are carried out usingwell-known procedures. For example, a variety of methods that may finduse in the present invention are described in Molecular Cloning—ALaboratory Manual, 3^(rd) Ed. (Maniatis, Cold Spring Harbor LaboratoryPress, New York, 2001), and Current Protocols in Molecular Biology (JohnWiley & Sons). As will be appreciated by those skilled in the art, thegeneration of exact sequences for a library comprising a large number ofsequences is potentially expensive and time consuming. Accordingly,there are a variety of techniques that may be used to efficientlygenerate libraries of the present invention. Such methods that may finduse in the present invention are described or referenced in U.S. Pat.No. 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S.Ser. No. 10/218,102; PCT WO 01/40091; and PCT WO 02/25588. Such methodsinclude but are not limited to gene assembly methods, PCR-based methodand methods which use variations of PCR, ligase chain reaction-basedmethods, pooled oligo methods such as those used in synthetic shuffling,error-prone amplification methods and methods which use oligos withrandom mutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode Fc variant members of a library.

The Fc variants of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the Fc variants, under theappropriate conditions to induce or cause expression of the protein. Theconditions appropriate for expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. A wide varietyof appropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in the present invention aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In a preferred embodiment, the Fc variants are expressed in mammalianexpression systems, including systems in which the expression constructsare introduced into the mammalian cells using virus such as retrovirusor adenovirus. Any mammalian cells may be used, with human, mouse, rat,hamster, and primate cells being particularly preferred. Suitable cellsalso include known research cells, including but not limited to Jurkat Tcells, NIH3T3, CHO, COS, and 293 cells. In an alternately preferredembodiment, library proteins are expressed in bacterial cells. Bacterialexpression systems are well known in the art, and include Escherichiacoil (E. coli), Bacillus subtilis, Streptococcus cremoris, andStreptococcus lividans. In alternate embodiments, Fc variants areproduced in insect cells or yeast cells. In an alternate embodiment, Fcvariants are expressed in vitro using cell free translation systems. Invitro translation systems derived from both prokaryotic (e.g. E. coli)and eukaryotic (e.g. wheat germ, rabbit reticulocytes) cells areavailable and may be chosen based on the expression levels andfunctional properties of the protein of interest. For example, asappreciated by those skilled in the art, in vitro translation isrequired for some display technologies, for example ribosome display. Inaddition, the Fc variants may be produced by chemical synthesis methods.

The nucleic acids that encode the Fc variants of the present inventionmay be incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria, insect cells, yeast, and in in vitro systems.As is known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing Fc variant proteins.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the Fc variant, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

Fc variants may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the Fc variant sequencevia a linker sequences. The linker sequence will generally comprise asmall number of amino acids, typically less than ten, although longerlinkers may also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs Fc variant protein and any associated fusion partners to adesired cellular location or to the extracellular media. As is known inthe art, certain signaling sequences may target a protein to be eithersecreted into the growth media, or into the periplasmic space, locatedbetween the inner and outer membrane of the cell. A fusion partner mayalso be a sequence that encodes a peptide or protein that enablespurification and/or screening. Such fusion partners include but are notlimited to polyhistidine tags (His-tags) (for example H₆ and H₁₀ orother tags for use with Immobilized Metal Affinity Chromatography (IMAC)systems (e.g. Ni⁺² affinity columns)), GST fusions, MBP fusions,Strep-tag, the BSP biotinylation target sequence of the bacterial enzymeBirA, and epitope tags which are targeted by antibodies (for examplec-myc tags, flag-tags, and the like). As will be appreciated by thoseskilled in the art, such tags may be useful for purification, forscreening, or both. For example, an Fc variant may be purified using aHis-tag by immobilizing it to a Ni⁺² affinity column, and then afterpurification the same His-tag may be used to immobilize the antibody toa Ni⁺² coated plate to perform an ELISA or other binding assay (asdescribed below). A fusion partner may enable the use of a selectionmethod to screen Fc variants (see below). Fusion partners that enable avariety of selection methods are well-known in the art, and all of thesefind use in the present invention. For example, by fusing the members ofan Fc variant library to the gene III protein, phage display can beemployed (Kay et al., Phage display of peptides and proteins: alaboratory manual, Academic Press, San Diego, Calif., 1996; Lowman etal., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science228:1315-1317). Fusion partners may enable Fc variants to be labeled.Alternatively, a fusion partner may bind to a specific sequence on theexpression vector, enabling the fusion partner and associated Fc variantto be linked covalently or noncovalently with the nucleic acid thatencodes them. For example, U.S. Ser. No. 09/642,574; U.S. Ser. No.10/080,376; U.S. Ser. No. 09/792,630; U.S. Ser. No. 10/023,208; U.S.Ser. No. 09/792,626; U.S. Ser. No. 10/082,671; U.S. Ser. No. 09/953,351;U.S. Ser. No. 10/097,100; U.S. Ser. No. 60/366,658; PCT WO 00/22906; PCTWO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO01/28702; and PCT WO 02/07466 describe such a fusion partner andtechnique that may find use in the present invention.

The methods of introducing exogenous nucleic acid into host cells arewell known in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In a preferred embodiment, Fc variant proteins are purified or isolatedafter expression. Proteins may be isolated or purified in a variety ofways known to those skilled in the art. Standard purification methodsinclude chromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of Fc variants. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies, as of course does the antibody's targetantigen. Purification can often be enabled by a particular fusionpartner. For example, Fc variant proteins may be purified usingglutathione resin if a GST fusion is employed, Ni⁺² affinitychromatography if a His-tag is employed, or immobilized anti-flagantibody if a flag-tag is used. For general guidance in suitablepurification techniques, see Protein Purification: Principles andPractice, 3^(rd) Ed., Scopes, Springer-Verlag, NY, 1994. The degree ofpurification necessary will vary depending on the screen or use of theFc variants. In some instances no purification is necessary. For examplein one embodiment, if the Fc variants are secreted, screening may takeplace directly from the media. As is well known in the art, some methodsof selection do not involve purification of proteins. Thus, for example,if a library of Fc variants is made into a phage display library,protein purification may not be performed.

Fc variants may be screened using a variety of methods, including butnot limited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners has been discussed above. By “labeled” herein is meantthat the Fc variants of the invention have one or more elements,isotopes, or chemical compounds attached to enable the detection in ascreen. In general, labels fall into three classes: a) immune labels,which may be an epitope incorporated as a fusion partner that isrecognized by an antibody, b) isotopic labels, which may be radioactiveor heavy isotopes, and c) small molecule labels, which may includefluorescent and colorimetric dyes, or molecules such as biotin thatenable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In a preferred embodiment, the functional and/or biophysical propertiesof Fc variants are screened in an in vitro assay. In vitro assays mayallow a broad dynamic range for screening properties of interest.Properties of Fc variants that may be screened include but are notlimited to stability, solubility, and affinity for Fc ligands, forexample FcγRs. Multiple properties may be screened simultaneously orindividually. Proteins may be purified or unpurified, depending on therequirements of the assay. In one embodiment, the screen is aqualitative or quantitative binding assay for binding of Fc variants toa protein or nonprotein molecule that is known or thought to bind the Fcvariant. In a preferred embodiment, the screen is a binding assay formeasuring binding to the antibody's or Fc fusions' target antigen. In analternately preferred embodiment, the screen is an assay for binding ofFc variants to an Fc ligand, including but are not limited to the familyof FcγRs, the neonatal receptor FcRn, the complement protein C1q, andthe bacterial proteins A and G. Said Fc ligands may be from anyorganism, with humans, mice, rats, rabbits, and monkeys preferred.Binding assays can be carried out using a variety of methods known inthe art, including but not limited to FRET (Fluorescence ResonanceEnergy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™ (Amplified Luminescent Proximity HomogeneousAssay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBIACORE®, isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the Fc variant. Assays may employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

The biophysical properties of Fc variant proteins, for example stabilityand solubility, may be screened using a variety of methods known in theart. Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, Fc variantproteins of the present invention may be unfolded using chemicaldenaturant, heat, or pH, and this transition may be monitored usingmethods including but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an Fc variant protein may bequantitatively or qualitatively determined using a wide range of methodsthat are known in the art. Methods which may find use in the presentinvention for characterizing the biophysical properties of Fc variantproteins include gel electrophoresis, chromatography such as sizeexclusion chromatography and reversed-phase high performance liquidchromatography, mass spectrometry, ultraviolet absorbance spectroscopy,fluorescence spectroscopy, circular dichroism spectroscopy, isothermaltitration calorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an Fc variant could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the Fc variant's stability and solubility.

In a preferred embodiment, the library is screened using one or morecell-based or in vivo assays. For such assays, Fc variant proteins,purified or unpurified, are typically added exogenously such that cellsare exposed to individual variants or pools of variants belonging to alibrary. These assays are typically, but not always, based on thefunction of an antibody or Fc fusion that comprises the Fc variant; thatis, the ability of the antibody or Fc fusion to bind a target antigenand mediate some biochemical event, for example effector function,ligand/receptor binding inhibition, apoptosis, and the like. Such assaysoften involve monitoring the response of cells to antibody or Fc fusion,for example cell survival, cell death, change in cellular morphology, ortranscriptional activation such as cellular expression of a natural geneor reporter gene. For example, such assays may measure the ability of Fcvariants to elicit ADCC, ADCP, or CDC. For some assays additional cellsor components, that is in addition to the target cells, may need to beadded, for example example serum complement, or effector cells such asperipheral blood monocytes (PBMCs), NK cells, macrophages, and the like.Such additional cells may be from any organism, preferably humans, mice,rat, rabbit, and monkey. Antibodies and Fc fusions may cause apoptosisof certain cell lines expressing the antibody's target antigen, or theymay mediate attack on target cells by immune cells which have been addedto the assay. Methods for monitoring cell death or viability are knownin the art, and include the use of dyes, immunochemical, cytochemical,and radioactive reagents. For example, caspase staining assays mayenable apoptosis to be measured, and uptake or release of radioactivesubstrates or fluorescent dyes such as alamar blue may enable cellgrowth or activation to be monitored. In a preferred embodiment, theDELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, MA) is used.Alternatively, dead or damaged target cells may be monitoried bymeasuring the release of one or more natural intracellular proteins, forexample lactate dehydrogenase. Transcriptional activation may also serveas a method for assaying function in cell-based assays. In this case,response may be monitored by assaying for natural genes or proteinswhich may be upregulated, for example the release of certaininterleukins may be measured, or alternatively readout may be via areporter construct. Cell-based assays may also involve the measure ofmorphological changes of cells as a response to the presence of an Fcvariant. Cell types for such assays may be prokaryotic or eukaryotic,and a variety of cell lines that are known in the art may be employed.

Alternatively, cell-based screens are performed using cells that havebeen transformed or transfected with nucleic acids encoding the Fcvariants. That is, Fc variant proteins are not added exogenously to thecells. For example, in one embodiment, the cell-based screen utilizescell surface display. A fusion partner can be employed that enablesdisplay of Fc variants on the surface of cells (Witrrup, 2001, Curr OpinBiotechnol, 12:395-399). Cell surface display methods that may find usein the present invention include but are not limited to display onbacteria (Georgiou et al., 1997, Nat Biotechnol 15:29-34; Georgiou etal., 1993, Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol18:645-648; June et al., 1998, Nat Biotechnol 16:576-80), yeast (Boder &Wittrup, 2000, Methods Enzymol 328:430-44; Boder & Wittrup, 1997, NatBiotechnol 15:553-557), and mammalian cells (Whitehorn et al., 1995,Biol-technology 13:1215-1219). In an alternate embodiment, Fc variantproteins are not displayed on the surface of cells, but rather arescreened intracellularly or in some other cellular compartment. Forexample, periplasmic expression and cytometric screening (Chen et al.,2001, Nat Biotechnol 19: 537-542), the protein fragment complementationassay (Johnsson & Varshavsky, 1994, Proc Natl Acad Sci USA91:10340-10344.; Pelletier et al., 1998, Proc Natl Acad Sci USA95:12141-12146), and the yeast two hybrid screen (Fields & Song, 1989,Nature 340:245-246) may find use in the present invention.Alternatively, if a polypeptide that comprises the Fc variants, forexample an antibody or Fc fusion, imparts some selectable growthadvantage to a cell, this property may be used to screen or select forFc variants.

As is known in the art, a subset of screening methods are those thatselect for favorable members of a library. Said methods are hereinreferred to as “selection methods”, and these methods find use in thepresent invention for screening Fc variant libraries. When libraries arescreened using a selection method, only those members of a library thatare favorable, that is which meet some selection criteria, arepropagated, isolated, and/or observed. As will be appreciated, becauseonly the most fit variants are observed, such methods enable thescreening of libraries that are larger than those screenable by methodsthat assay the fitness of library members individually. Selection isenabled by any method, technique, or fusion partner that links,covalently or noncovalently, the phenotype of an Fc variant with itsgenotype, that is the function of an Fc variant with the nucleic acidthat encodes it. For example the use of phage display as a selectionmethod is enabled by the fusion of library members to the gene IIIprotein. In this way, selection or isolation of variant proteins thatmeet some criteria, for example binding affinity for an FcγR, alsoselects for or isolates the nucleic acid that encodes it. Once isolated,the gene or genes encoding Fc variants may then be amplified. Thisprocess of isolation and amplification, referred to as panning, may berepeated, allowing favorable Fc variants in the library to be enriched.Nucleic acid sequencing of the attached nucleic acid ultimately allowsfor gene identification.

A variety of selection methods are known in the art that may find use inthe present invention for screening Fc variant libraries. These includebut are not limited to phage display (Phage display of peptides andproteins: a laboratory manual, Kay et al., 1996, Academic Press, SanDiego, Calif., 1996; Lowman et al., 1991, Biochemistry 30:10832-10838;Smith, 1985, Science 228:1315-1317) and its derivatives such asselective phage infection (Malmborg et al., 1997, J Mol Biol273:544-551), selectively infective phage (Krebber et al., 1997, J MolBiol 268:619-630), and delayed infectivity panning (Benhar et al., 2000,J Mol Biol 301:893-904), cell surface display (Witrrup, 2001, Curr OpinBiotechnol, 12:395-399) such as display on bacteria (Georgiou et al.,1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993, Trends Biotechnol11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; Jun et al., 1998,Nat Biotechnol 16:576-80), yeast (Boder & Wittrup, 2000, Methods Enzymol328:430-44; Boder & Wittrup, 1997, Nat Biotechnol 15:553-557), andmammalian cells (Whitehorn et al., 1995, Bioltechnology 13:1215-1219),as well as in vitro display technologies (Amstutz et al., 2001, CurrOpin Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes etal., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts &Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302; Nemoto et al.,1997, FEBS Lett 414:405-408), and ribosome-inactivation display system(Zhou et al., 2002, J Am Chem Soc 124, 538-543)

Other selection methods that may find use in the present inventioninclude methods that do not rely on display, such as in vivo methodsincluding but not limited to periplasmic expression and cytometricscreening (Chen et al., 2001, Nat Biotechnol 19:537-542), the proteinfragment complementation assay (Johnsson & Varshavsky, 1994, Proc NatlAcad Sci USA 91:10340-10344; Pelletier et al., 1998, Proc Natl Acad SciUSA 95:12141-12146), and the yeast two hybrid screen (Fields & Song,1989, Nature 340:245-246) used in selection mode (Visintin et al., 1999,Proc Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,selection is enabled by a fusion partner that binds to a specificsequence on the expression vector, thus linking covalently ornoncovalently the fusion partner and associated Fc variant librarymember with the nucleic acid that encodes them. For example, U.S. Ser.No. 09/642,574; U.S. Ser. No. 10/080,376; U.S. Ser. No. 09/792,630; U.S.Ser. No. 10/023,208; U.S. Ser. No. 09/792,626; U.S. Ser. No. 10/082,671;U.S. Ser. No. 09/953,351; U.S. Ser. No. 10/097,100; U.S. Ser. No.60/366,658; PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466 describesuch a fusion partner and technique that may find use in the presentinvention. In an alternative embodiment, in vivo selection can occur ifexpression of a polypeptide that comprises the Fc variant, such as anantibody or Fc fusion, imparts some growth, reproduction, or survivaladvantage to the cell.

A subset of selection methods referred to as “directed evolution”methods are those that include the mating or breading of favorablesequences during selection, sometimes with the incorporation of newmutations. As will be appreciated by those skilled in the art, directedevolution methods can facilitate identification of the most favorablesequences in a library, and can increase the diversity of sequences thatare screened. A variety of directed evolution methods are known in theart that may find use in the present invention for screening Fc variantlibraries, including but not limited to DNA shuffling (PCT WO 00/42561A3; PCT WO 01/70947 A3), exon shuffling (U.S. Pat. No. 6,365,377;Kolkman & Stemmer, 2001, Nat Biotechnol 19:423-428), family shuffling(Crameri et al., 1998, Nature 391:288-291; U.S. Pat. No. 6,376,246),RACHITT™ (Coco et al., 2001, Nat Biotechnol 19:354-359; PCT WO02/06469), STEP and random priming of in vitro recombination (Zhao etal., 1998, Nat Biotechnol 16:258-261; Shao et al., 1998, Nucleic AcidsRes 26:681-683), exonuclease mediated gene assembly (U.S. Pat. Nos.6,352,842; 6,361,974), Gene Site Saturation Mutagenesis™ (U.S. Pat. No.6,358,709), Gene Reassembly™ (U.S. Pat. No. 6,358,709), SCRATCHY (Lutzet al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA fragmentationmethods (Kikuchi et al., Gene 236:159-167), single-stranded DNAshuffling (Kikuchi et al., 2000, Gene 243:133-137), and AMEsystem™directed evolution protein engineering technology (Applied MolecularEvolution) (U.S. Pat. Nos. 5,824,514; 5,817,483; 5,814,476; 5,763,192;5,723,323).

The biological properties of the antibodies and Fc fusions that comprisethe Fc variants of the present invention may be characterized in cell,tissue, and whole organism experiments. As is know in the art, drugs areoften tested in animals, including but not limited to mice, rats,rabbits, dogs, cats, pigs, and monkeys, in order to measure a drug'sefficacy for treatment against a disease or disease model, or to measurea drug's pharmacokinetics, toxicity, and other properties. Said animalsmay be referred to as disease models. Therapeutics are often tested inmice, including but not limited to nude mice, SCID mice, xenograft mice,and transgenic mice (including knockins and knockouts). For example, anantibody or Fc fusion of the present invention that is intended as ananti-cancer therapeutic may be tested in a mouse cancer model, forexample a xenograft mouse. In this method, a tumor or tumor cell line isgrafted onto or injected into a mouse, and subsequently the mouse istreated with the therapeutic to determine the ability of the antibody orFc fusion to reduce or inhibit cancer growth. Such experimentation mayprovide meaningful data for determination of the potential of saidantibody or Fc fusion to be used as a therapeutic. Any organism,preferably mammals, may be used for testing. For example because oftheir genetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the antibodies and Fc fusions ofthe present invention. Tests of the antibodies and Fc fusions of thepresent invention in humans are ultimately required for approval asdrugs, and thus of course these experiments are contemplated. Thus theantibodies and Fc fusions of the present invention may be tested inhumans to determine their therapeutic efficacy, toxicity,pharmacokinetics, and/or other clinical properties.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

For all positions discussed in the present invention, numbering isaccording to the EU index as in Kabat (Kabat et al., 1991, Sequences ofProteins of Immunological Interest, 5th Ed., United States Public HealthService, National Institutes of Health, Bethesda). Those skilled in theart of antibodies will appreciate that this convention consists ofnonsequential numbering in specific regions of an immunoglobulinsequence, enabling a normalized reference to conserved positions inimmunoglobulin families. Accordingly, the positions of any givenimmunoglobulin as defined by the EU index will not necessarilycorrespond to its sequential sequence. FIG. 3 shows the sequential andEU index numbering schemes for the antibody alemtuzumab in order toillustrate this principal more clearly. It should also be noted thatpolymorphisms have been observed at a number of Fc positions, includingbut not limited to Kabat 270, 272, 312, 315, 356, and 358, and thusslight differences between the presented sequence and sequences in thescientific literature may exist.

Example 1 Computational Screening and Design of Fc Libraries

Computational screening calculations were carried out to designoptimized Fc variants. Fc variants were computationally screened,constructed, and experimentally investigated over severalcomputation/experimention cycles. For each successive cycle,experimental data provided feedback into the next set of computationalscreening calculations and library design. All computational screeningcalculations and library design are presented in Example 1. For each setof calculations, a table is provided that presents the results andprovides relevant information and parameters.

Several different structures of Fc bound bound to the extracellulardomain of FcγRs served as template structures for the computationalscreening calculations. Publicly available Fc/FcγR complex structuresincluded pdb accession code 1E4K (Sondermann et al., 2000, Nature406:267-273.), and pdb accession codes 1IIS and 1IIX (Radaev et al.,2001, J Biol Chem 276:16469-16477). The extracellular regions ofFcγRIIIb and FcγRIIIa are 96% identical, and therefore the use of theFc/FcγRIIIb structure is essentially equivalent to use of FcγRIIIa.Nonetheless, for some calculations, a more precise Fc/FcγRIIIa templatestructure was constructed by modeling a D129G mutation in the 1IIS and1E4K structures (referred to as D129G 1IIS and D129G 1E4K templatestructures). In addition, the structures for human Fc bound to theextracellular domains of human FcγRIIb, human F158 FcγRIIIa, and mouseFcγRIII were modeled using standard methods, the available FcγR sequenceinformation, the aforementioned Fc/FcγR structures, as well asstructural information for unbound complexes (pdb accession code1H9V)(Sondermann et al. 2001, J Mol Biol 309:737-749) (pdb accessioncode 1FCG)(Maxwell et al., 1999, Nat Struct Biol 6:437-442), FcγRIIb(pdb accession code 2FCB)(Sondermann et al., 1999, Embo J 18:1095-1103),and FcγRIIIb (pdb accession code 1E4J)(Sondermann et al., 2000, Nature406:267-273.).

Variable positions and amino acids to be considered at those positionswere chosen by visual inspection of the aforementioned Fc/FcγR and FcγRstructures, and using solvent accessibility information and sequenceinformation. Sequence information of Fcs and FcγRs was particularlyuseful for determining variable positions at which substitutions mayprovide distinguishing affinities between activating and inhibitoryreceptors. Virtually all Cγ2 positions were screened computationally.The Fc structure is a homodimer of two heavy chains (labeled chains Aand B in the 1IIS, 1IIX, and 1E4K structures) that each include thehinge and Cγ2-Cγ3 domains (shown in FIG. 2). Because the FcγR (labeledchain C in the 1IIS, 1IIX, and 1E4K structures) binds asymmetrically tothe Fc homodimer, each chain was often considered separately in designcalculations. For some calculations, Fc and/or FcγR residues proximal tovariable position residues were floated, that is the amino acidconformation but not the amino acid identity was allowed to vary in aprotein design calculation to allow for conformational adjustments.These are indicated below the table for each set of calculations whenrelevant. Considered amino acids typically belonged to either the Core,Core XM, Surface, Boundary, Boundary XM, or All 20 classifications,unless noted otherwise. These classifications are defined as follows:Core=alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,tryptophan, and methionine; Core XM=alanine, valine, isoleucine,leucine, phenylalanine, tyrosine, and tryptophan; Surface=alanine,serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid,arginine, lysine and histidine; Boundary=alanine, serine, threonine,aspartic acid, asparagine, glutamine, glutamic acid, arginine, lysine,histidine, valine, isoleucine, leucine, phenylalanine, tyrosine,tryptophan, and methionine; Boundary XM=Boundary=alanine, serine,threonine, aspartic acid, asparagine, glutamine, glutamic acid,arginine, lysine, histidine, valine, isoleucine, leucine, phenylalanine,tyrosine, and tryptophan; All 20 =all 20 naturally occurring aminoacids.

The majority of calculations followed one of two general types ofcomputational screening methods. In one method, the conformations ofamino acids at variable positions were represented as a set ofbackbone-independent side chain rotamers derived from the rotamerlibrary of Dunbrack & Cohen (Dunbrack et al., 1997, Protein Sci6:1661-1681). The energies of all possible combinations of theconsidered amino acids at the chosen variable positions were calculatedusing a force field containing terms describing van der Waals,solvation, electrostatic, and hydrogen bond interactions, and theoptimal (ground state) sequence was determined using a Dead EndElimination (DEE) algorithm. As will be appreciated by those in the art,the predicted lowest energy sequence is not necessarily the true lowestenergy sequence because of errors primarily in the scoring function,coupled with the fact that subtle conformational differences in proteinscan result in dramatic differences in stability. However, the predictedground state sequence is likely to be close to the true ground state,and thus additional favorable diversity can be explored by evaluatingthe energy of sequences that are close in sequence space and energyaround the predicted ground state. To accomplish this, as well as togenerate a diversity of sequences for a library, a Monte Carlo (MC)algorithm was used to evaluate the energies of 1000 similar sequencesaround the predicted ground state. The number of sequences out of the1000 sequence set that contain that amino acid at that variable positionis referred to as the occupancy for that substitution, and this valuemay reflect how favorable that substitution is. This computationalscreening method is substantially similar to Protein Design Automation®(PDA®) technology, as described in U.S. Pat. Nos. 6,188,965; 6,269,312;6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser.No. 10/218,102; PCT WO 98/07254; PCT WO 01/40091; and PCT WO 02/25588,and for ease of description, is referred to as PDA® technologythroughout the examples. Tables that present the results of thesecalculations provide for each variable position on the designated chain(column 1) the amino acids considered at each variable position (column2), the WT Fc amino acid identity at each variable position (column 3),the amino acid identity at each variable position in the DEE groundstate sequence (column 4), and the set of amino acids and correspondingoccupancy that are observed in the Monte Carlo output (column 5). Forexample in the first row of Table 1 below, when position 328 was variedusing boundary amino acids as the set of variable residues for thatposition, L occurred 330 times in the top 1000 sequence, M occurred 302times, etc.

Other calculations utilized a genetic algorithm (GA) to screen for lowenergy sequences, with energies being calculated during each round of“evolution” for those sequences being sampled. The conformations ofamino acids at variable and floated positions were represented as a setof side chain rotamers derived from a backbone-independent rotamerlibrary using a flexible rotamer model (Mendes et al. 1999, Proteins37:530-543). Energies were calculated using a force field containingterms describing van der Waals, solvation, electrostatic, and hydrogenbond interactions. This calculation generated a list of 300 sequenceswhich are predicted to be low in energy. To facilitate analysis of theresults and library generation, the 300 output sequences were clusteredcomputationally into 10 groups of similar sequences using a nearestneighbor single linkage hierarchical clustering algorithm to assignsequences to related groups based on similarity scores (Diamond, 1995,Acta Cryst D51 :127-135). That is, all sequences within a group are mostsimilar to all other sequences within the same group and less similar tosequences in other groups. The lowest energy sequence from each of theseten clusters are used as a representative of each group, and arepresented as results. This computational screening method issubstantially similar to Sequence Prediction Algorithm™ (SPA™)technology, as described in (Raha et al., 2000, Protein Sci9:1106-1119); U.S. Ser. No. 09/877,695; and U.S. Ser. No. 10/071,859,and for ease of description, is referred to as SPA™ technologythroughout the examples. Tables that present the results of thesecalculations provide for each variable position on the designated chain(column 1) the amino acids considered at each variable position (column2), the WT Fc amino acid identity at each variable position (column 3),and the amino acid identity at the variable positions for the lowestenergy sequence from each cluster group (columns 4-13).

Computational screening was applied to design energetically favorableinteractions at the Fc/FcγR interface at groups of variable positionsthat mediate or potentially mediate binding with FcγR. Because thebinding interface involves a large number of Fc residues on the twodifferent chains, and because FcγRs bind asymmetrically to Fc, residueswere grouped in different sets of interacting variable positions, anddesigned in separate sets of calculations. In many cases these sets werechosen as groups of residues that were deemed to be coupled, that is theenergy of one or more residues is dependent on the identity of one ormore other residues. Various template structures were used, and in manycases calculations explored substitutions on both chains. For many ofthe variable position sets, calculations were carried out using both thePDA®) and SPA™ technology computational screening methods described. Theresults of these calculations and relevant are presented in Tables 1-30below. Relevant parameters and information are presented below eachtable, including the computational screening method used, the templatestructure used, whether or not that structure had carbohydrate atoms,and any residues that may have been floated. For example, Table 2presents results from a PDA® calculation in which residues 120, 132, and134 on chain C (the FcγRIIIb receptor) were floated.

Included within the compositions of the invention are antibodies thathave any of the listed amino acid residues in the listed positions,either alone or in any combination (note preferred combinations arelisted in the claims, the summary and the figures). One preferredcombination is the listed amino acid residues in the listed positions ina ground state (sometimes referred to herein as the “global solution”,as distinguished from the wild-type). In addition, combinations betweenSPA™ proteins, both within tables and between tables, are also included.It should be noted that residues not listed in a given table are impliedto have not been varied, and thus remain wild-type. For example, in theSPA™ calculation results presented in Table 4, column 4 (representingcluster 1) indicates a protein with the six listed amino acids at thesix listed positions (e.g. column 4 is a single protein with a wild-typesequence except for 239E, 265G, 267S, 269Y, 270T and 299S). Thus, eachof these individual proteins are included within the invention.Alternatively, residue positions and particular amino acids at thoseresidue positions may be combined between columns within a table, orbetween tables. Furthermore, it should be noted that although each tableindicates the presence or absence of carbohydrate, the presence orabsence of said atoms in the computational screening calculation is notmeant to imply that Fc variants designed by such calculations should beapplicable to only aglycosylated or glycosylated Fc. Thus although thecalculations in Table 1 were run without carbohydrate atoms present inthe template structure, the resulting predicted substitutions may befavorable in a glycosylated or aglycosylated antibody or Fc fusion.TABLE 1 Considered Ground Sequences Around Position Amino Acids WT StateGround State 328 A Boundary L L L:330 M:302 E:111 K:62 A:45 Q:39 D:36S:30 T:28 N:10 R:7 332 A Surface I R R:247 K:209 Q:130 H:95 E:92 T:59D:51 N:51 S:42 A:24 328 B Boundary L L L:321 M:237 T:166 K:73 R:72 S:55Q:20 D:17 E:13 A:12 V:10 N:4 332 B Surface I E E:269 Q:180 R:145 K:111D:97 T:78 N:65 S:28 A:14 H:13

PDA® technology, 1IIS template structure; −carbohydrate TABLE 2Considered Ground Sequences Around Position Amino Acids WT State GroundState 239 A Surface S K E:349 D:203 K:196 A:95 Q:83 S:63 N:10 R:1 265 ABoundary XM D D D:616 N:113 L:110 E:104 S:25 A:23 Q:9 299 A Boundary XMT I I:669 H:196 V:135 327 A Boundary XM A S A:518 S:389 N:67 D:26 265 BBoundary XM D Q Q:314 R:247 N:118 I:115 A:63 E:55 D:34 S:22 K:21 V:11

PDA® technology; 1IIS template structure; +carbohydrate; floated 120 C,132 C, 134 C TABLE 3 Considered Ground Sequences Around Position AminoAcids WT State Ground State 239 A Surface S E E:872 Q:69 D:39 K:16 A:4265 A Boundary XM D Y Y:693 H:111 E:69 D:62 F:29 K:19 R:14 W:2 Q:1 267 ABoundary XM S S S:991 A:9 269 A Core XM E F F:938 E:59 Y:3 270 A SurfaceD E E:267 T:218 K:186 D:89 Q:88 R:46 S:34 N:29 H:23 A:20 299 A BoundaryXM T H H:486 T:245 K:130 E:40 S:39 D:27 Q:27 A:4 N:2

PDA® technology; 1IIS template structure; −carbohydrate; floated 120 C,122 C, 132 C, I33 C, 134 C TABLE 4 Considered Position Amino Acids WT 12 3 4 5 6 7 8 9 10 239 A Surface S E Q Q Q E E E Q E E 265 A All 20 D GG G G G G G G G G 267 A All 20 S S S S S S S S S S S 269 A Core E Y Y AA V Y A A A A 270 A Surface D T S A S T T T A A A 299 A All 20 T S S S SS S S S S S

SPA™ technology; 1IIS template structure; +carbohydrate; floated 120 C,122 C, 132 C, I33 C, 134 C TABLE 5 Considered Ground Sequences AroundPosition Amino Acids WT State Ground State 235 A Boundary XM L T T:195V:131 L:112 W:107 K:85 F:66 Y:56 E:52 Q:38 S:37 I:34 R:29 H:26 N:23 D:9296 A Surface Y N N:322 D:181 R:172 K:76 Y:70 Q:59 E:48 S:40 H:20 T:11A:1 298 A Surface S T T:370 R:343 K:193 A:55 S:39 235 B Boundary XM L LL:922 I:78

PDA® technology; 1IIS template structure; −carbohydrate; floated 119 C,128 C, 157 C TABLE 6 Considered Position Amino Acids WT 1 2 3 4 5 6 7 89 10 235 A All 20 L S S P S S S S S S S 296 A Surface Y Q Q Q E E Q E QQ N 298 A Surface S S K K K K S S S K S 235 B All 20 L K K K L L L L L LK

SPA™ technology; 1IIS template structure; +carbohydrate; floated 119 C,128 C, 157 C TABLE 7 Considered Ground Sequences Around Position AminoAcids WT State Ground State 239 B Surface S E K:402 E:282 H:116 T:67R:47 Q:39 D:26 A:11 S:7 N:3 265 B Boundary XM D W Y:341 W:283 I:236 V:77F:36 H:9 T:7 E:4 K:4 A:2 D:1 327 B Boundary XM A R R:838 K:86 H:35 E:12T:10 Q:7 A:6 D:3 N:3 328 B Core XM L L L:1000 329 B Core XM P P P:801A:199 330 B Core XM A Y Y:918 F:42 L:22 A:18 332 B Surface I I I:792E:202 Q:5 K:1

PDA® technology; 1IIS template structure; −carbohydrate; floated 88 C,90 C, 113 C, 114 C, 116 C, 160 C, 161 C TABLE 8 Considered PositionAmino Acids WT 1 2 3 4 5 6 7 8 9 10 239 B Surface S D T E E E E E E E E265 B All 20 D G G K G K G G K K G 327 B All 20 A K M L L N L K L L L328 B Core L M M M L A M L M L L 329 B Core P P P P P P P P P P P 330 BCore A L A A A A A A A A A 332 B Surface I I Q I I Q Q E D I I

SPA™ technology; 1IIS template structure; +carbohydrate; floated 88 C,90 C, 113 C, 114 C, 116 C, 160 C, 161 C TABLE 9 Considered PositionAmino Acids WT 1 2 3 4 5 6 7 8 9 10 239 A Surface S Q Q Q E Q E Q E Q Q265 A All 20 D G G G G G G G G G G 299 A All 20 T S S A S S S S S S S327 A All 20 A A S S S S S S S A S 265 B All 20 D N G G G G G G G G G

SPA™ technology; 1IIS template structure; −carbohydrate; floated 120 C,132 C, 134 C TABLE 10 Considered Ground Sequences Around Position AminoAcids WT State Ground State 234 A Boundary XM L K Y:401 L:260 F:151 I:82K:63 H:17 Q:11 W:7 R:3 T:2 E:2 V:1 235 A Boundary XM L L W:777 L:200K:12 Y:5 I:3 F:2 V:1 234 B Boundary XM L W W:427 Y:203 L:143 F:74 I:59E:32 K:23 V:14 D:10 T:7 H:4 R:4 235 B Boundary XM L W W:380 Y:380 F:135K:38 L:26 E:15 Q:12 H:8 R:4 T:2

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated113 C, 116 C, 132 C, 155 C, 157C TABLE 11 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 234 A All 20 L G G G G G G G G G G 235 AAll 20 L T L L L L L L L T L 234 B All 20 L G G G G G G G G G G 235 BAll 20 L S A S A A S S S A A

SPA™ technology; D129G 1E4K template structure; +carbohydrate; floated113 C, 116 C, 132 C, 155 C, 157 C TABLE 12 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 A Boundary XM S EE:235 S:122 D:94 Q:93 A:74 K:70 L:67 T:63 N:57 R:51 I:29 V:18 W:15 H:12328 A Boundary XM L L L:688 E:121 K:43 Q:41 A:33 D:26 S:14 T:14 N:12 R:8332 A Boundary XM I W I:155 W:95 L:82 K:79 E:74 Q:69 H:67 V:63 R:57 T:57D:45 S:43 N:42 A:35 F:19 Y:18

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated120 C TABLE 13 Considered Position Amino Acids WT 1 2 3 4 5 6 7 8 9 10239 A All 20 S L E E Q E E K K K K 328 A All 20 L L Q L Q K L L Q K L332 A All 20 I K K L Q A K L Q A Q

SPA™ technology; D129G 1IIS template structure; +carbohydrate; floated120 C TABLE 14 Considered Ground Sequences Around Position Amino AcidsWT State Ground State 239 B Boundary XM S I R:195 I:169 L:126 V:91 K:89E:61 H:52 T:50 Q:42 N:35 S:34 D:30 A:26 328 B Boundary XM L L L:671T:165 K:40 S:38 E:28 R:17 Q:17 V:11 A:8 D:5 332 B Boundary XM I I I:387E:157 L:151 V:78 Q:63 K:50 R:33 T:29 D:25 A:12 N:8 S:6 W:1

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated90 C, 160 C, 161 C TABLE 15 Considered Position Amino Acids WT 1 2 3 4 56 7 8 9 10 239 B All 20 S T L L L L L L L L L 328 B All 20 L M R M D T ML Q D L 332 B All 20 I I D Q Q Q L L T Q L

SPA™ technology; D129G 1IIS template structure; +carbohydrate; floated90 C, 160 C, 161 C TABLE 16 Considered Ground Sequences Around PositionAmino Acids WT State Ground State 239 B Boundary XM S T T:164 S:159L:156 E:86 W:76 K:71 D:65 A:52 R:43 H:38 Q:38 N:31 I:14 V:7 328 BBoundary XM L L L:556 E:114 T:84 K:80 S:69 Q:36 A:31 D:15 R:11 N:4 332 BBoundary XM I W I:188 W:177 E:97 L:94 T:59 Q:57 V:54 K:52 F:51 D:34 H:33S:27 R:26 N:18 A:17 Y:16

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated117 C TABLE 17 Considered Position Amino Acids WT 1 2 3 4 5 6 7 8 9 10239 B All 20 S P S P E L L L L L L 328 B All 20 L K K K K K L K K K L332 B All 20 I S S E L L L E L L L

SPA™ technology; D129G 1E4K template structure; +carbohydrate; floated117 C TABLE 18 Considered Ground Sequences Around Position Amino AcidsWT State Ground State 239 A Boundary XM S L K:196 L:171 I:146 E:88 V:76R:75 T:50 H:45 D:43 Q:39 S:30 N:22 A:19 328 A Boundary XM L W L:517F:230 W:164 H:40 K:29 E:11 R:5 T:4 332 A Boundary XM I E I:283 L:217E:178 Q:81 V:64 D:47 T:35 K:27 W:18 R:12 A:10 Y:7 N:7 F:6 S:5 H:3

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated87 C, 157 C, 158 C TABLE 19 Considered Position Amino Acids WT 1 2 3 4 56 7 8 9 10 239 A All 20 S F Q E T P P T P P P 328 A All 20 L K R R K K MR K M R 332 A All 20 I L L I I E I E E I I

SPA™ technology; D129G 1E4K template structure; +carbohydrate atoms;floated 87 C, 157 C, 158 C TABLE 20 Considered Ground Sequences AroundPosition Amino Acids WT State Ground State 240 A Core + Thr V V V:698M:162 T:140 263 A Core + Thr V V V:966 T:34 266 A Core + Thr V V V:983T:17 325 A Boundary N N N:943 T:40 A:17 328 A Boundary L L L:610 M:363K:27 332 A Glu I E E:1000

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated273 A, 275 A, 302 A, 323 A, 134C TABLE 21 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 240 A All 20 V V A V V V V V V V V 263 AAll 20 V V V V V V V V V V V 266 A All 20 V I V I I T V V V V I 325 AAll 20 N A N N N Q T T Q N T 328 A All 20 L K K L K L K L L L L 332 AGlu I D D D D D D D D D D

SPA™ technology; D129G 1IIS template structure; +carbohydrate; floated273 A, 275 A, 302 A, 323 A, 134C TABLE 22 Considered Ground SequencesAround Position Amino Acids WT State Ground State 240 B Core + Thr V VV:713 T:287 263 B Core + Thr V V V:992 T:8 266 B Core + Thr V V V:976T:24 325 B Boundary N N N:453 T:296 A:116 D:96 S:30 V:9 328 B Boundary LL L:623 M:194 T:100 R:72 K:11 332 B Glu I E E:1000

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated273 B, 275 B, 302 B, 323 B, 161 C TABLE 23 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 240 B All 20 V A T A T T A A T T T 263 BAll 20 V V A A T T V V T A T 266 B All 20 V V V V V V V V V I V 325 BAll 20 N N K K N K K N N N N 328 B All 20 L R L L L L L L L L L 332 AGlu I D D D D D D D D D D

SPA™ technology; D129G 1IIS template structure; +carbohydrate; floated273 B, 275 B, 302 B, 323 B, 161 C TABLE 24 Considered Ground SequencesAround Position Amino Acids WT State Ground State 240 B Core + Thr V MV:715 M:271 T:12 I:2 263 B Core + Thr V V V:992 T:8 266 B Core + Thr V VV:996 T:4 325 B Boundary N N N:651 T:232 D:64 A:53 328 B Boundary L MM:556 L:407 K:37 332 B Glu I E E:1000

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated273 B, 275 B, 302 B, 323 B, 131 C TABLE 25 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 240 B All 20 V T A T A A A A T A A 263 BAll 20 V T W T T A T T T L L 266 B All 20 V L A T T V L T T L V 325 BAll 20 N A N A A N A A A A A 328 B All 20 L L K L L L L L L L L 332 AGlu I D D D D D D D D D D

SPAT technology; D129G 1E4K template structure; +carbohydrate; floated273 B, 275 B, 302 B, 323 B, 131 C TABLE 26 Considered Ground SequencesAround Position Amino Acids WT State Ground State 240 A Core + Thr V VV:876 T:109 M:15 263 A Core + Thr V V V:913 T:87 266 A Core + Thr V VV:969 T:31 325 A Boundary N V V:491 N:236 T:187 A:35 D:32 S:19 328 ABoundary L L L:321 W:290 M:271 F:49 K:46 R:23 332 A Glu I E E:1000

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated273 A, 275 A, 302 A, 323 A, 158 C TABLE 27 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 240 A All 20 V A T A A T T A A A T 263 AAll 20 V T T V V T V L L V T 266 A All 20 V V V V V V V V V V V 325 AAll 20 N Q N Q Q Q Q Q Q N N 328 A All 20 L K M K K K K K K K K 332 AGlu I D D D D D D D D D DSPA™ technology; D129G 1E4K template structure; +carbohydrate; floated273 A, 275 A, 302 A, 323 A, 158C

Computational screening calculations were aimed at designing Fc variantsto optimize the conformation of the N297 carbohydrate and the Cγ2domain. By exploring energetically favorable substitutions at positionsthat interact with carbohydrate, variants can be engineered that samplenew, potentially favorable carbohydrate conformations. Fc residues F241,F243, V262, and V264 mediate the Fc/carbohydrate interaction and thusare target positions. The results of these design calculations arepresented in Table 28. TABLE 28 Considered Ground Sequences AroundPosition Amino Acids WT State Ground State 241 A Core F Y Y:172 M:162L:144 F:140 W:110 I:97 A:91 V:84 243 A Core F Y Y:211 L:204 W:199 F:160M:141 A:85 262 A Core V M M:302 I:253 V:243 A:202 264 A Core V F I:159M:152 V:142 L:140 W:136 F:120 Y:104 A:47PDA® technology, 1IIS template structure; −carbohydrate

Computational screening calculations were aimed at designing Fc variantsto optimize the angle between the Cγ3 and Cγ2 domains. Residues P244,P245, P247, and W313, which reside at the Cγ2/Cγ3 interface, appear toplay a key role in determining the Cγ2-Cγ3 angle and the flexibility ofthe domains relative to one another. By exploring energeticallyfavorable substitutions at these positions, variants can be designedthat sample new, potentially favorable angles and levels of flexibility.The results of these design calculations are presented in Table 29.TABLE 29 Considered Ground Sequences Around Position Amino Acids WTState Ground State 244 A Boundary P H K:164 H:152 R:110 M:100 S:92 N:57A:54 D:50 Q:49 T:46 E:37 V:30 L:27 W:23 F:9 245 A Boundary P A A:491S:378 N:131 247 A Boundary P V V:156 T:125 K:101 E:87 Q:79 R:78 S:76A:72 D:72 H:60 M:47 N:47 313 A Boundary W W W:359 F:255 Y:128 M:114 H:48K:29 T:24 A:11 E:10 V:10 S:9 Q:3PDA® technology; 1IIS template structure; −carbohydrate

In addition to the above calculations using PDA® and SPA™ computationalscreening methods, additional calculations using solely an electrostaticpotential were used to computationally screen Fc variants. Calculationswith Coulomb's law and Debye-Huckel scaling highlighted a number ofpositions in the Fc for which amino acid substitutions would favorablyaffect binding to one or more FcγRs, including positions for whichreplacement of a neutral amino acid with a negatively charged amino acidmay enhance binding to FcγRIIIa, and for which replacement of apositively charged amino acid with a neutral or negatively charged aminoacid may enhance binding to FcγRIIIa. These results are presented inTable 30. TABLE 30 Replacement of a + residue Replacement of a neutralresidue with a − residue with a − residue H268 S239 K326 Y296 K334 A327I332Coulomb's law and Debye-Huckel scaling; 1IIS template structure;+carbohydrate

Computational screening calculations were carried out to optimizeaglycosylated Fc, that is to optimize Fc structure, stability,solubility, and Fc/FcγR affinity in the absence of the N297carbohydrate. Design calculations were aimed at designing favorablesubstitutions in the context of the aglycosylated Fc template structureat residue 297, residues proximal to it, residues at the Fc/FcγRinterface, and residues at the Fc/carbohydrate interface. Variablepositions were grouped in different sets of interacting variablepositions and designed in separate sets of calculations, and varioustemplate structures were used. For many of the variable position sets,calculations were carried out using both the PDA® and SPA™ computationalscreening methods. The results of these calculations and relevantinformation are presented in Tables 31-53 below. TABLE 31 ConsideredGround Sequences Around Position Amino Acids WT State Ground State 265 ABoundary XM D Y Y:531 F:226 W:105 H:92 K:21 D:16 E:6 T:3 297 A BoundaryXM N D A:235 S:229 D:166 E:114 N:92 Y:57 F:55 Q:25 H:10 T:7 K:6 L:3 R:1299 A Boundary XM T L L:482 Y:186 F:131 T:55 S:51 K:31 H:22 A:18 E:14Q:10 297 B Boundary XM N I I:299 K:147 V:85 R:82 W:71 N:65 D:35 E:35Q:34 S:32 L:31 H:30 T:28 A:26

PDA® technology; 1IIS template structure; −carbohydrate; floated 122 C,129 C, 132 C, 155 C TABLE 32 Considered Position Amino Acids WT 1 2 3 45 6 7 8 9 10 265 A All 20 D G G G G G G G G G G 297 A All 20 N A T A E KK A A N N 299 A All 20 T S K S K F F F F F S 297 B All 20 N K K K K K KK K K K

SPA™ technology; 1IIS template structure; −carbohydrate; floated 122 C,129 C, 132 C, 155 C TABLE 33 Considered Ground Sequences Around PositionAmino Acids WT State Ground State 239 A Surface S E E:928 Q:65 D:7 265 ABoundary XM D W W:709 Y:248 F:43 296 A Surface Y H H:449 Y:146 E:137D:89 K:64 N:32 T:30 R:25 Q:23 S:5 297 A Surface N E E:471 H:189 D:102T:97 K:96 R:22 Q:15 S:8 298 A Boundary XM S R R:353 T:275 K:269 A:56S:38 E:5 Q:2 H:2 299 A Boundary XM T F Y:398 F:366 L:217 H:15 K:4

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 128 C, 132 C, 155 C TABLE 34 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 A All 20 S E Q Q E Q Q Q Q Q Q 265 AAll 20 D G G G G G G G G G G 296 A All 20 Y D Q N N Q N N N Q N 297 AAll 20 N A A N A D D E N N E 298 A All 20 S K K K S K K K K S K 299 AAll 20 T S Y F S Y F K F S K

SPA™ technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 128 C, 132 C, 155 C TABLE 35 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 B Surface S EE:417 T:122 D:117 Q:94 R:84 S:63 K:47 H:29 N:19 A:8 265 B Boundary XM DW W:865 Y:79 F:55 K:1 296 B Surface Y Y Y:549 H:97 D:80 S:75 N:48 E:45K:32 R:30 Q:28 A:16 297 B Surface N R R:265 H:224 E:157 K:154 Q:75 D:47T:34 N:24 S:13 A:7 298 B Boundary XM S V V:966 D:10 T:8 A:8 N:4 S:4 299B Boundary XM T Y Y:667 F:330 H:3

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 125 C, 129 C, 152 C TABLE 36 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 B All 20 S S R E K S S E E E K 265 BAll 20 D A D K Y A A F F K Y 296 B All 20 Y A A A A A A A A A A 297 BAll 20 N T S T T E E E S E E 298 B All 20 S G G G G G G G G G G 299 BAll 20 T L F E E Y F Y F Y Y

SPA™ technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 125 C, 129 C, 152 C TABLE 37 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 A Surface S EE:868 Q:92 D:38 K:1 N:1 265 A Boundary XM D W W:575 Y:343 F:66 H:15 K:1296 A Surface Y H H:489 Y:103 R:98 K:97 Q:64 D:63 T:41 N:38 E:7 297 AAsp N D D:1000 298 A Boundary XM S R R:340 K:262 T:255 A:59 S:57 E:11Q:10 H:6 299 A Boundary XM T F Y:375 F:323 L:260 H:24 K:18

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 128 C, 132 C, 155 C TABLE 38 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 A All 20 S E Q E E E E E E Q E 265 AAll 20 D G G G G G G G G G G 296 A All 20 Y E N Q E N Q Q Q Q N 297 AAsp N D D D D D D D D D D 298 A All 20 S K S K S K K K S K K 299 A All20 T S K Y S F F F F F K

SPA™ technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 128 C, 132 C, 155 C TABLE 39 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 B Surface S EE:318 Q:123 T:109 D:108 R:93 S:89 K:69 N:40 H:38 A:13 265 B Boundary XMD W W:745 Y:158 F:85 K:9 E:1 R:1 H:1 296 B Surface Y Y Y:390 H:127 S:83R:81 K:78 N:65 D:55 E:49 Q:44 A:26 T:2 297 B Asp N D D:1000 298 BBoundary XM S V V:890 T:35 A:29 D:19 S:16 N:10 E:1 299 B Boundary XM T YY:627 F:363 H:10

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 125 C, 129 C, 152C TABLE 40 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 B All 20 S K E E Q E K Q E K Q 265 BAll 20 D F K K A K Y W K L F 296 B All 20 Y A A A A A A A A A A 297 BAsp N D D D D D D D D D D 298 B All 20 S G G G G G G G G G G 299 B All20 T Y Y Y Y Y Y F F Y Y

SPA™ technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 125 C, 129 C, 152 C TABLE 41 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 A Boundary XM S EE:312 L:148 D:102 Q:98 K:64 I:61 S:57 A:44 T:39 N:29 R:23 V:18 W:5 265 ABoundary XM D W W:363 Y:352 F:139 H:77 K:39 R:14 D:11 E:4 Q:1 297 A AspN D D:1000 299 A Boundary XM T Y Y:309 F:224 L:212 H:96 K:92 E:28 Q:20R:16 T:2 S:1

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 132 C, 155 C TABLE 42 Considered Position Amino Acids WT 12 3 4 5 6 7 8 9 10 239 A All 20 S E L L L E E E Q L E 265 A All 20 D G GG G G G G G G G 297 B Asp N D D D D D D D D D D 299 A All 20 T S K K F FF K F K F

SPA™ technology; D129G 1IIS template structure; −carbohydrate; floated120 C, 122 C, 132 C, 155C TABLE 43 Considered Ground Sequences AroundPosition Amino Acids WT State Ground State 239 B Boundary XM S L L:194T:122 S:120 E:111 D:79 K:71 A:62 Q:57 R:43 H:43 N:37 I:24 W:24 V:13 265B Boundary XM D W Y:248 W:233 F:198 K:84 D:57 E:55 H:42 R:28 Q:20 A:10T:10 N:8 S:7 297 B Asp N D D:1000 299 B Boundary XM T Y Y:493 F:380 H:76T:31 E:10 D:4 A:3 S:3

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 129 C, 152 C TABLE 44 Considered Position Amino Acids WT 12 3 4 5 6 7 8 9 10 239 B All 20 S R E P L L F P P L L 265 B All 20 D D KS F S Y A M A D 297 B Asp N D D D D D D D D D D 299 B All 20 T Y Y Y Y EY Y Y Y Y

SPA™ technology; D129G 1E4K template structure; −carbohydrate; floated117 C, 119 C, 129 C, 152 C TABLE 45 Considered Ground Sequences AroundPosition Amino Acids WT State Ground State 239 A Boundary XM S E E:251L125 D:120 Q:112 S:73 K:65 I:61 A:58 T:45 N:35 R:28 V:23 W:4 265 ABoundary XM D Y Y:216 H:153 K:135 D:109 W:104 F:86 R:54 T:38 E:29 Q:22A:21 N:17 S:13 L:3 297 A Asp N D D:1000

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated299 A, 120 C, 122 C, 132 C, 155 C TABLE 46 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 A All 20 S S L E L Q Q E Q Q E 265 AAll 20 D G G G G G G G G G G 297 A Asp N D D D D D D D D D D

SPA™ technology; D129G 1IIS template structure; −carbohydrate; floated299 A, 120 C, 122 C, 132 C, 155 C TABLE 47 Considered Ground SequencesAround Position Amino Acids WT State Ground State 239 B Boundary XM S LL:158 S:137 T:125 E:115 D:86 K:75 A:62 Q:56 H:43 R:39 N:35 W:30 I:24V:15 265 B Boundary XM D Y Y:188 W:159 F:156 D:122 K:77 E:71 H:61 Q:44R:39 A:24 S:22 N:19 T:18 297 B Asp N D D:1000

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated299 B, 117 C, 119 C, 129 C, 152C TABLE 48 Considered Position AminoAcids WT 1 2 3 4 5 6 7 8 9 10 239 B All 20 S S E P P E S P L F L 265 BAll 20 D A K A M K F Y D F F 297 B Asp N D D D D D D D D D D

SPA™ technology; D129G 1E4K template structure; −carbohydrate; floated299 B, 117 C, 119 C, 129 C, 152 C TABLE 49 Considered Ground SequencesAround Position Amino Acids WT State Ground State 297 A Asp N D D:1000299 A Boundary XM T Y T:123 Y:64 H:64 K:64 Q:64 F:64 R:63 D:63 E:63 S:63L:63 N:62 I:57 A:54 V:52 W:17

PDA® technology; D129G 1IIS template structure; −carbohydrate; floated239 A, 265 A, 120 C, 122 C, 132 C, 155 C TABLE 50 Considered PositionAmino Acids WT 1 2 3 4 5 6 7 8 9 10 297 A Asp N D D D D D D D D D D 299A All 20 T K K K K F F K K K K

SPA™ technology; D129G 1IIS template structure; −carbohydrate; floated239 A, 265 A, 120 C, 122 C, 132 C, 155 C TABLE 51 Considered GroundSequences Around Position Amino Acids WT State Ground State 297 B Asp ND D:1000 299 B Boundary XM T Y T:123 F:64 Y:64 H:64 S:63 N:61 Q:61 D:61E:60 K:58 V:57 A:57 R:54 I:52 L:51 W:50

PDA® technology; D129G 1E4K template structure; −carbohydrate; floated239 B, 265 B, 117 C, 119C, 129 C, 152 C TABLE 52 Considered PositionAmino Acids WT 1 2 3 4 5 6 7 8 9 10 297 B Asp N D D D D D D D D D D 299B All 20 T Y Y Y Y Y Y Y Y Y YSPA™ technology; D129G 1E4K template structure; −carbohydrate; floated239 B, 265 B, 117 C, 119 C, 129 C, 152 C

Computational screening calculations were carried out to optimizeaglycosylated Fc by designing favorable substitutions at residues thatare exposed to solvent in the absence of glycosylation such that theyare stable, maintain Fc structure, and have no tendency to aggregate.The N297 carbohydrate covers up the exposed hydrophobic patch that wouldnormally be the interface for a protein-protein interaction with anotherIg domain, maintaining the stability and structural integrity of Fc andkeeping the Cγ2 domains from aggregating across the central axis. Keyresidues for design are F241, F243, V262, and V264, which reside behindthe carbohydrate on Cγ2, in addition to residues such as L328, I332, andI336, which are exposed nonpolar residues facing inward towards theopposed Cγ2 domain, that were considered in previously presentedcalculations. The importance of these Cγ2 residues is supported bynoting that the corresponding residues in the Cγ3 domain by sequencealignment either mediate the nonpolar interaction between the two Cγ3domains or are buried in the Cγ3 core. The results of these designcalculations are presented in Table 53. TABLE 53 Considered GroundSequences Around Position Amino Acids WT State Ground State 241 ASurface F E E:190 R:172 K:138 H:117 T:93 Q:91 D:85 S:49 N:49 A:16 243 ASurface F R R:190 H:164 Q:152 E:149 K:92 T:71 D:64 N:58 S:42 A:18 262 ASurface V D D:416 E:164 N:138 Q:87 T:83 R:44 S:32 K:24 A:11 H:1 264 ASurface V H R:368 H:196 K:147 E:108 Q:68 T:34 N:33 D:25 S:15 A:6PDA® technology; 1IIS template structure; −carbohydrate

In a final set of calculations, a SPA™ computational screening methodwas applied to evaluate the replacement of all chosen variable positionswith all 20 amino acids. The lowest energy rotamer conformation for all20 amino acids was determined, and this energy was defined as the energyof substitution for that amino acid at that variable position. Thesecalculations thus provided an energy of substitution for each of the 20amino acids at each variable position. The calculations used varioustemplate structures including different Fc/FcγRIIIb complexes (1IIS,1IIX, 1E4K), a modeled Fc/FcγRIIb complex, and uncomplexed Fc (1DN2),and thus were useful for a variety of design goals aimed at bothglycosylated and aglycosylated Fc, including optimization of Fc/FcγRaffinity, C1q affinity, Fc stability, Fc solubility, carbohydrateconformation, and hinge conformation. Furthermore, because thesecalculations provide energies for both favorable and unfavorablesubstitutions, they guide substitutions that may enable differentialbinding to activating versus inhibitory FcγRs. Various templatestructures were used, and calculations explored substitutions on bothchains. The results of these calculations and relevant parameters andinformation are presented in Tables 54-60 below. Column 1 lists thevariable positions on chain A and B of the template structure. Column 2lists the wild-type amino acid identity at each variable position. Theremaining 20 columns provide the energy for each of the natural 20 aminoacids (shown in the top row). All substitutions were normalized withrespect to the lowest energy substitution, which was set to 0 energy.For example in Table 54, for L235 on chain A, serine is the lowestenergy substitution, and L235A is 0.9 kcal/mol less stable than L235S.Extremely high energies were set to 20 kcal/mol for energies between20-50 kcal/mol, and 50 kcal/mold for energies greater than 50 kcal/mol.Favorable substitutions may be considered to be the lowest energysubstitution for each position, and substitutions that have small energydifferences from the lowest energy substitution, for examplesubstitutions within 1-2, 1-3, 1-5, or 1-10 kcal/mol. TABLE 54 Pos WT AC D E F G H I K L M 235 A L 0.9 2.8 2.8 1.5 3.2 3.2 3.4 4.9 1.6 2.1 3.2236 A G 0 1.9 5.1 6.7 10 2.3 4.3 17.2 5.7 20 4.6 237 A G 20 20 20 50 500 50 50 20 50 20 239 A S 0.2 4.3 2.6 0 12.8 4.5 6.9 11.3 1.7 0.1 2.1 265A D 9.0 8.1 6.3 7.8 5.1 0 7.3 50 8.2 9.9 7.7 267 A S 2.1 3.3 7.3 1.4 507.3 20 20 0.9 2.2 5.0 269 A E 0.5 2.1 1.3 0.6 1.6 3.9 2.0 1.2 1.1 1.32.7 270 A D 0.3 2.8 2.3 2.0 4.0 4.0 3.4 2.4 1.2 0 2.3 296 A Y 2.7 2.01.4 0 50 0 50 4.6 2.1 2.4 3.3 298 A S 0.7 2.4 6.7 3.4 20 3.9 20 6.7 04.1 1.4 299 A T 0.6 2.8 11.5 10.1 20 6.1 20 10.7 7.1 20 4.3 234 B L 2.13.2 4.1 4.2 1.6 5.3 0.1 0.7 0.6 1.0 2.0 235 B L 0.6 2.3 2.5 0.7 5.4 4.81.4 3.6 0.1 0 2.0 236 B G 3.1 1.3 4.4 8.2 5.2 0 1.9 20 3.1 20 4.1 237 BG 20 50 50 50 50 0 50 50 50 50 50 239 B S 0.9 2.4 3.4 1.8 5.4 5.6 2.73.0 0.9 0 2.0 265 B D 4.5 5.1 4.6 4.6 4.9 0 3.8 9.0 2.0 2.5 4.1 327 B A1.8 3.4 4.7 3.9 20 7.0 20 20 0.8 0 1.9 328 B L 3.7 3.6 4.0 3.7 50 8.46.8 50 3.8 0 2.1 329 B P 3.4 8.6 20 20 50 8.0 16.8 50 20 20 16.9 330 B A0.5 2.0 2.6 0.5 2.4 3.8 1.4 4.2 0 2.0 2.2 332 B I 1.5 2.7 1.2 1.6 11.96.8 12.9 1.2 2.9 0 1.4 Pos WT N P Q R S T V W Y 235 A L 0.9 0.3 1.3 0.70 1.7 4.3 6.5 3.2 236 A G 3.2 12.6 5.6 6.1 0.6 6.2 12.0 6.7 20 237 A G20 50 50 50 20 20 50 50 50 239 A S 1.7 7.9 1.2 2.6 0.3 5.7 11.0 20 20265 A D 6.0 50 9.0 8.5 7.8 20 50 20 5.8 267 A S 4.8 0 2.2 3.1 2.9 20 2050 50 269 A E 0 50 0.6 1.1 0.3 0.8 1.0 5.6 1.2 270 A D 2.1 20 2.0 2.31.4 1.8 4.2 5.4 6.0 296 A Y 1.2 50 0.2 1.5 1.3 4.6 4.4 16.3 18.2 298 A S4.1 50 1.8 1.1 0.2 2.2 6.3 17.8 20 299 A T 6.8 50 6.3 12.0 0 3.0 7.114.8 20 234 B L 1.7 50 2.8 0.3 2.3 1.7 2.6 13.0 0 235 B L 1.7 16.6 0.51.2 0.7 0.7 5.3 6.8 5.5 236 B G 2.7 50 3.7 16.0 1.2 20 20 20 11.3 237 BG 50 50 50 50 50 50 50 50 50 239 B S 1.6 50 1.8 1.8 1.4 1.4 5.1 20 5.3265 B D 2.1 50 4.5 5.1 4.4 5.9 9.2 11.4 5.8 327 B A 1.5 20 3.0 2.6 3.220 20 20 20 328 B L 4.1 50 3.6 8.1 4.9 3.0 12.5 50 50 329 B P 20 0 20 201.3 17.1 16.5 50 50 330 B A 0.8 20 0.1 0.6 0.9 0.3 5.1 8.0 2.7 332 B I1.7 50 1.3 4.9 1.8 1.7 3.0 20 20

SPA™ technology; 1IIS template structure; +carbohydrate atoms, nofloated positions TABLE 55 Pos WT A C D E F G H I K L M 235 A L 0.9 2.82.6 1.7 3.3 3.3 3.4 5.0 1.6 2.1 3.3 236 A G 0 1.7 5.2 6.0 11.3 2.3 4.417.2 5.8 19.0 4.9 237 A G 20 20 20 50 50 0 50 50 20 50 20 238 A P 8.68.0 10.5 13.4 6.4 0 5.0 50 12.4 11.3 9.7 239 A S 0.1 4.2 2.5 0 20 4.59.0 10.8 1.8 0.2 2.1 240 A V 1.3 2.4 2.3 6.3 20 7.2 20 5.1 10.8 6.2 5.7241 A F 0.1 1.6 1.2 0.3 0.2 4.1 1.2 10 1.3 0.1 2.1 242 A L 3.0 3.4 5.58.3 14.4 8.5 11.1 3.3 13.9 2.2 2.7 243 A F 1.6 2.2 2.7 0.2 1.4 5.6 2.5 02.2 2.0 3.0 244 A P 1.2 1.8 3.8 0.8 10.2 3.8 4.6 20 0.2 2.9 2.0 245 A P3.9 20 20 20 20 9.1 20 20 20 20 20 246 A K 1.3 2.7 2.0 2.0 2.9 5.7 2.91.4 1.4 1.5 3.1 247 A P 1.2 2.1 0.3 0.7 4.0 3.9 3.7 1.8 1.6 1.7 3.3 248A K 0.9 2.7 1.5 0.8 3.1 4.7 3.4 3.3 2.0 1.9 2.6 249 A D 1.2 3.7 1.6 0 207.3 19.7 50 1.7 20 2.2 250 A T 0 1.8 3.8 5.8 50 6.0 20 4.5 6.3 6.3 0.3251 A L 1.1 1.9 1.2 0.5 5.8 5.1 1.9 5.6 0.9 0.7 2.4 252 A M 0.3 1.2 0.60 3.0 3.8 3.4 3.9 1.0 0.3 2.2 253 A I 0.7 1.7 1.1 0.2 1.8 3.5 2.2 2.00.3 1.2 0.8 254 A S 0.7 1.7 0.4 0.7 2.2 3.6 2.0 0.3 1.2 1.9 2.4 255 A R1.4 2.8 2.4 2.5 0.2 5.4 1.1 17.0 1.0 2.2 1.5 256 A T 0.6 1.8 1.2 1.1 2.73.4 2.1 1.4 0.7 1.5 2.4 257 A P 0 7.8 20 12.9 50 6.2 50 20 12.3 12.814.4 258 A E 0 1.6 4.8 2.6 1.0 4.3 2.2 14.8 4.4 6.2 3.2 259 A V 3.9 4.35.1 8.7 20 10.3 6.8 2.3 9.6 2.8 6.2 260 A T 1.7 2.3 3.3 1.1 20 6.6 8.6 00.2 1.8 2.8 261 A C 0 20 20 20 20 3.9 20 20 20 20 20 262 A V 1.9 3.2 03.3 20 7.2 20 8.3 2.9 2.9 2.2 263 A V 2.2 2.7 6.0 17.4 20 8.8 20 10 7.17.6 16.9 264 A V 1.9 3.3 2.8 2.2 0 6.4 2.1 0.7 2.6 0.9 2.7 265 A D 9.08.1 5.9 8.6 5.3 0 7.3 50 7.9 9.7 7.5 266 A V 4.9 5.3 7.1 12.1 20 11.2 200.4 12.2 20 8.8 267 A S 2.3 3.5 7.2 1.3 50 7.4 20 20 0.7 1.4 3.9 268 A H1.2 1.9 2.2 1.5 3.7 5.0 4.9 0.4 0.5 3.7 2.7 269 A E 0.3 1.9 1.3 0.5 1.33.7 1.9 1.1 0.8 1.2 2.5 270 A D 0.2 2.6 2.1 1.9 5.2 3.9 3.1 2.1 1.2 02.2 271 A P 0 5.3 8.1 9.3 20 3.1 9.1 20 6.0 9.5 5.3 272 A Q 0.8 1.9 0.91.2 3.0 3.2 3.7 3.7 1.6 1.8 3.2 273 A V 1.2 2.9 1.8 20 20 7.1 20 6.8 2020 20 274 A K 0.4 1.8 1.4 0.8 1.9 3.9 2.4 1.4 0.7 1.1 2.9 275 A F 8.09.5 10.3 9.5 0 13.5 5.1 10.1 6.2 6.3 6.0 276 A N 1.3 2.4 2.4 2.2 0.8 5.10.8 1.2 0.6 2.3 2.5 277 A W 5.5 7.4 8.4 6.4 15.4 11.2 3.2 8.2 1.9 3.93.6 278 A Y 1.6 2.7 3.9 1.6 1.0 7.3 3.4 17.7 1.4 7.5 2.1 279 A V 3.1 4.14.0 2.2 20 8.1 9.7 8.5 0 1.4 3.1 280 A D 1.8 2.6 2.7 0.2 11.5 2.9 8.8 203.4 3.2 2.8 281 A G 50 50 50 50 50 0 50 50 50 50 50 282 A V 0.9 2.1 1.61.1 2.9 4.2 3.5 1.4 1.5 1.8 3.6 283 A E 0.7 1.6 0.7 0.5 1.0 4.4 1.4 0.41.2 1.8 1.9 284 A V 0 2.2 3.1 1.2 20 5.0 20 4.0 0.7 2.6 0.8 285 A H 0.21.4 3.1 1.3 3.0 2.0 2.4 3.6 1.1 2.6 3.0 286 A N 0.8 2.5 1.2 1.1 2.4 4.72.7 2.1 0 0.7 1.8 287 A A 0.6 2.6 5.8 3.3 10.4 5.4 9.1 11.3 0 4.4 1.3288 A K 0.8 2.6 2.0 1.3 3.0 3.4 3.8 2.3 1.4 1.7 2.5 289 A T 0.3 1.9 4.71.1 3.1 3.6 2.9 10.5 0.4 2.7 1.6 290 A K 1.7 2.2 0.5 0.6 3.0 1.3 3.0 3.71.7 2.1 3.2 291 A P 1.6 3.1 1.8 0.5 1.9 5.5 1.8 0.1 0.5 1.5 1.2 292 A R1.1 2.2 3.1 0.8 5.9 4.4 8.0 5.0 0 1.6 2.1 293 A E 2.2 6.5 9.0 17.9 16.30 13.2 50 12.8 10.3 10.3 294 A E 1.5 2.1 2.1 0.7 8.1 2.8 3.3 2.0 2.6 1.82.8 295 A Q 50 50 50 50 50 0 50 50 50 50 50 296 A Y 2.8 2.3 1.1 0.4 50 050 4.6 2.2 2.3 3.1 297 A N 0 6.5 8.4 5.3 20 3.4 20 13.8 2.7 20 4.8 298 AS 0.8 2.4 5.7 2.2 20 3.7 20 6.2 0.9 9.2 1.8 299 A T 1.9 3.4 6.0 3.1 1.07.1 2.9 3.1 0 2.7 2.6 300 A Y 2.8 2.9 2.7 4.5 20 4.0 7.5 13.1 1.2 0 2.2301 A R 3.0 3.5 3.8 2.8 0.8 3.4 1.8 0 1.3 0.7 2.6 302 A V 2.7 4.6 6.73.9 2.8 8.9 1.2 6.9 2.7 2.0 2.2 303 A V 0 2.2 3.3 1.0 6.7 4.5 5.3 1.42.5 3.1 2.0 304 A S 0 12.1 10.8 20 20 6.2 20 20 17.2 20 11.9 305 A V 1.12.3 3.3 1.2 0.3 5.4 1.2 0 0.9 1.1 2.8 306 A L 4.3 6.2 7.1 5.9 2.8 10.43.4 13.7 3.0 0 3.5 307 A T 1.4 3.2 3.8 2.2 6.5 5.5 4.2 0.5 0.3 4.2 3.0308 A V 1.8 5.5 6.5 8.0 50 7.9 20 4.5 20 5.5 19.4 309 A L 1.1 2.7 0.70.7 1.3 4.6 2.7 0.7 1.7 1.0 2.8 310 A H 2.0 2.6 0.9 4.1 50 5.6 0.2 6.84.0 7.1 4.0 311 A Q 0.6 2.5 1.6 1.6 2.5 4.3 1.6 1.4 0.6 0.9 2.9 312 A N5.4 5.1 5.9 1.3 20 0 20 10 3.4 4.8 3.3 313 A W 4.6 6.4 5.5 5.6 1.1 10.85.0 11.0 5.8 5.2 7.6 314 A L 2.1 2.9 4.3 2.2 5.7 6.1 7.9 5.4 0.7 0 1.7315 A D 0.3 1.4 1.5 0.1 3.3 4.2 1.9 1.8 0.8 0.5 1.8 316 A G 50 50 50 5050 0 50 50 50 50 50 317 A K 0 14.0 18.4 17.9 50 5.0 50 20 8.5 12.5 12.7318 A E 2.0 3.0 2.7 1.7 2.7 6.7 2.6 0 1.1 1.6 1.3 319 A Y 2.9 4.4 3.93.4 0 8.8 1.8 20 0.5 5.2 0.7 320 A K 2.3 3.1 3.0 2.7 20 7.8 20 9.4 0 0.62.7 321 A C 0 3.2 20 18.8 20 6.9 20 20 20 20 10.4 322 A K 2.0 2.5 3.52.8 2.7 6.4 2.1 0.2 0.1 1.2 2.7 323 A V 1.5 2.8 7.3 11.9 20 8.1 20 6.09.6 20 4.9 324 A S 2.0 2.1 0.6 0 1.9 4.9 3.9 1.5 2.8 0.7 1.9 325 A N 2.83.9 8.4 3.0 20 8.3 20 0 7.7 20 6.2 326 A K 1.0 2.7 3.0 1.6 3.7 4.1 3.13.2 1.7 2.4 3.7 327 A A 0.9 2.8 5.8 3.1 20 6.3 16.7 14.7 2.8 20 2.5 328A L 6.0 6.3 7.0 4.1 50 8.6 20 50 5.7 0 7.1 329 A P 1.0 2.5 0.9 0.6 4.03.4 3.3 1.7 1.9 2.5 3.6 330 A A 0.9 2.0 1.3 0.7 3.4 3.8 3.0 2.0 1.4 2.03.4 331 A P 50 50 50 50 50 0 50 50 50 50 50 332 A I 1.9 3.7 4.6 1.7 5.07.0 1.9 3.8 1.8 0 2.5 333 A E 0 3.1 3.2 0.8 4.1 4.4 4.2 16.9 3.6 2.8 2.8334 A K 1.7 2.9 2.5 0 1.0 6.1 3.3 1.0 1.5 0.5 3.5 335 A T 0.5 3.2 4.52.7 4.2 4.9 4.1 20 2.1 3.1 3.0 336 A I 1.2 1.6 5.0 1.5 20 6.1 16.8 0.73.4 7.8 2.5 337 A S 4.8 4.8 7.5 11.5 10.1 0 5.5 50 9.9 7.0 7.9 338 A K1.0 2.7 2.3 2.2 4.6 5.9 2.4 50 0 2.1 1.9 339 A A 1.0 2.5 0.8 1.1 4.4 3.73.7 2.1 1.8 2.6 3.6 340 A K 1.3 2.4 2.3 2.0 1.7 4.1 2.3 1.9 0 2.3 1.8232 B P 1.3 3.2 2.2 2.2 4.1 2.9 3.6 1.8 2.1 2.8 3.9 233 B E 0.5 2.2 1.70.5 2.6 3.7 2.9 4.4 1.4 1.1 3.2 234 B L 2.9 4.0 4.8 4.9 2.0 6.1 0.8 1.50 1.9 2.7 235 B L 0.6 2.3 2.4 0.9 5.7 4.9 1.4 3.7 0 0 1.9 236 B G 3.62.5 5.1 11.8 6.8 0 2.8 20 5.0 20 4.5 237 B G 20 50 50 50 50 0 50 50 5050 50 238 B P 3.5 4.7 8.5 4.2 20 9.8 20 0 5.6 9.6 4.6 239 B S 1.0 2.53.4 2.0 7.2 5.7 3.1 3.1 0.6 0 2.0 240 B V 0.1 2.3 7.0 11.9 20 6.5 20 8.112.7 20 12.0 241 B F 0 2.0 1.4 0.8 1.0 4.0 2.0 6.5 1.1 0.6 2.3 242 B L2.2 3.3 6.5 6.6 6.9 7.9 4.3 0 8.7 3.9 4.8 243 B F 0.8 2.6 1.9 1.7 0.84.9 2.0 3.6 1.2 0.8 2.5 244 B P 1.1 2.1 4.0 1.1 11.9 3.5 5.4 20 1.4 3.23.0 245 B P 3.2 20 20 20 20 8.6 20 20 20 20 20 246 B K 0.5 2.6 1.4 1.22.1 4.4 1.6 0.6 0.9 1.4 2.5 247 B P 0.8 2.5 0.7 1.0 3.6 3.9 2.6 6.2 1.82.1 2.9 248 B K 0.2 2.2 0.2 0.6 2.2 4.1 2.5 2.4 1.7 1.0 2.2 249 B D 2.83.3 0 4.6 10.1 8.2 6.5 50 4.6 6.2 4.4 250 B T 0 2.2 4.9 2.8 20 6.3 202.2 4.3 3.2 3.0 251 B L 0 2.4 1.6 1.2 5.6 3.6 2.2 7.4 1.2 0.6 2.3 252 BM 1.3 2.4 0.8 0 1.8 5.7 2.3 0.6 1.6 0.6 2.5 253 B I 1.6 3.0 2.0 1.2 3.74.5 3.5 2.9 0.8 2.4 2.4 254 B S 1.0 1.5 0.8 0.6 3.8 3.8 3.2 0.5 1.9 2.53.1 255 B R 0.9 2.0 2.0 1.7 0 5.4 1.4 20 1.0 1.6 1.3 256 B T 0.6 2.0 1.81.1 2.5 3.7 1.9 1.6 1.0 1.4 2.2 257 B P 2.5 20 20 20 50 9.0 50 20 20 2020 258 B E 1.5 2.4 2.7 1.4 2.7 6.4 4.2 0 0.2 5.4 2.4 259 B V 2.9 4.2 6.35.2 20 9.3 20 0 8.1 8.9 5.5 260 B T 0 1.6 5.3 1.9 20 4.9 20 0.6 1.1 2.81.4 261 B C 0 10 20 20 20 2.6 20 20 20 20 20 262 B V 2.1 2.4 2.7 2.4 8.17.2 3.8 1.8 3.5 8.6 3.4 263 B V 2.2 3.7 4.7 11.2 20 9.1 20 15.0 13.7 2.820 264 B V 2.1 3.0 4.6 2.7 8.6 6.8 6.6 0 1.8 1.8 3.7 265 B D 4.5 5.2 4.84.7 5.0 0 3.8 8.5 1.8 2.6 4.1 266 B V 5.3 5.5 7.2 12.7 20 12.0 20 2.1 2020 20 267 B S 2.8 4.3 6.2 3.8 0 7.4 1.0 50 1.0 0.3 3.2 268 B H 2.6 3.75.1 4.1 4.9 6.0 1.8 2.6 0 2.5 3.8 269 B E 0.4 2.4 1.7 0.8 2.8 3.7 2.61.0 1.0 1.6 3.0 270 B D 0 1.6 1.1 7.3 4.8 4.3 2.6 20 3.8 14.5 3.8 271 BP 1.1 3.3 5.6 3.4 4.1 5.5 4.2 20 1.9 3.6 3.9 272 B Q 0.9 1.9 1.0 0.6 3.03.9 2.9 1.5 1.7 2.2 3.5 273 B V 3.5 4.8 6.2 8.3 20 9.2 20 4.6 8.4 3.13.5 274 B K 0.1 1.6 0.4 0.9 1.7 3.8 1.8 1.9 0.4 0.5 2.4 275 B F 5.7 7.08.4 9.2 0 11.2 3.5 9.2 7.9 5.7 5.1 276 B N 0 6.2 6.9 6.4 20 4.7 12.1 209.3 10 7.4 277 B W 8.3 10 10.6 9.2 2.6 14.2 7.4 12.7 6.7 7.4 6.4 278 B Y0 2.3 17.4 4.0 50 5.1 50 20 2.8 20 2.1 279 B V 3.1 3.5 4.2 2.9 20 8.513.9 0.4 0 2.9 2.0 280 B D 0.5 3.0 2.1 1.5 6.7 3.1 4.7 12.6 2.9 1.6 2.9281 B G 5.6 5.8 5.5 4.8 7.9 0 7.2 6.5 5.3 5.7 7.1 282 B V 0.4 1.9 1.10.6 2.9 4.1 2.1 1.3 1.0 1.4 2.9 283 B E 0.6 1.9 4.3 1.7 6.7 4.2 5.2 2.90.5 4.4 0.3 284 B V 0.4 2.4 2.5 1.1 20 5.9 20 1.1 1.2 6.2 0.8 285 B H1.3 2.4 2.1 1.7 2.4 3.4 1.2 1.8 0.7 2.3 2.7 286 B N 1.2 2.7 1.0 1.1 3.03.1 2.6 0.8 2.0 1.9 2.9 287 B A 2.5 4.4 6.1 7.5 0 8.2 3.0 10.2 5.1 16.54.5 288 B K 0.4 1.9 1.9 0 2.9 3.5 2.9 2.5 1.8 2.1 2.5 289 B T 0.1 1.53.7 1.4 2.7 3.9 2.6 1.8 0 2.2 1.6 290 B K 0.9 1.8 0.8 0.5 2.4 0.8 2.73.0 1.3 1.3 2.6 291 B P 1.2 2.1 2.5 0.5 3.9 4.6 3.4 0.7 0 3.4 1.5 292 BR 0.8 2.6 3.3 1.2 4.9 3.6 6.8 3.1 2.0 2.4 2.2 293 B E 0 3.0 4.1 2.8 7.33.6 5.8 5.8 2.6 4.5 3.2 294 B E 2.5 3.3 3.9 2.3 8.3 6.8 4.4 5.6 3.6 2.33.7 295 B Q 1.1 2.2 1.9 0.6 3.8 2.8 3.1 8.0 1.4 2.2 3.4 296 B Y 1.5 2.71.2 1.2 4.1 4.1 3.5 1.1 1.8 2.7 3.5 297 B N 3.9 4.5 10.1 6.0 15.5 7.316.7 6.6 0 5.1 4.6 298 B S 1.7 2.5 3.5 2.5 2.5 3.7 2.4 3.0 0 1.8 2.3 299B T 0 2.7 7.2 11.1 20 4.8 20 7.5 6.9 20 7.1 300 B Y 3.8 5.2 8.0 4.3 208.6 20 12.2 0 4.3 3.2 301 B R 1.2 1.8 2.3 1.1 20 5.8 11.3 5.2 0.3 5.02.0 302 B V 3.5 4.8 5.5 3.7 0.2 9.6 1.1 0.5 2.6 3.5 2.5 303 B V 0.2 00.1 1.0 20 5.0 13.3 5.1 1.7 10.4 1.9 304 B S 1.5 2.3 8.2 20 20 7.6 207.6 20 20 20 305 B V 0.1 1.2 3.3 1.1 20 4.6 20 3.2 1.1 11.0 1.5 306 B L4.7 6.8 6.3 4.3 10.4 11.1 7.8 4.2 3.0 0 3.8 307 B T 1.5 30 2.7 1.7 4.15.2 3.0 1.6 1.9 3.1 3.4 308 B V 0 0.6 7.6 20 20 6.6 20 20 16.1 15.1 20309 B L 1.4 3.0 2.2 1.1 3.0 6.0 3.5 20 2.4 1.7 3.6 310 B H 2.4 2.9 2.74.9 20 6.8 4.4 4.8 3.1 15.0 3.4 311 B Q 0 2.2 1.3 0.7 2.1 3.3 2.4 12.60.6 0.9 2.3 312 B N 0 1.0 0.2 03 6.0 5.4 2.3 12.0 2.1 2.9 1.6 313 B W5.3 6.6 7.3 5.4 0 11.4 6.2 20 4.0 5.2 4.3 314 B L 1.7 2.2 3.1 0 6.4 5.61.5 2.1 0.6 0.2 1.1 315 B D 1.4 2.3 2.4 0.7 6.0 5.5 2.3 4.8 2.2 1.0 2.9316 B G 50 50 50 50 50 0 50 50 50 50 50 317 B K 0.9 2.3 4.3 2.8 1.2 4.00.6 13.9 0 4.8 1.6 318 B E 0.7 1.2 3.1 1.0 7.0 5.1 8.2 0.4 1.0 5.7 1.7319 B Y 6.5 7.1 8.5 8.8 0 12.5 3.9 3.1 5.2 5.4 8.4 320 B K 3.1 4.3 7.34.3 20 8.6 15.0 1.4 0 11.6 3.6 321 B C 0 6.5 20 20 20 6.6 20 20 20 20 20322 B K 2.3 3.2 3.5 1.8 20 7.9 20 1.1 0.6 4.9 3.7 323 B V 4.0 4.6 6.98.1 20 10.6 20 9.0 17.1 7.9 8.1 325 B N 3.4 5.1 9.0 4.7 20 8.2 20 16.616.6 20 20 326 B K 0.3 2.1 2.0 0.9 1.0 3.5 2.0 2.9 0.9 2.9 2.8 327 B A1.9 3.3 4.7 3.5 20 7.0 20 20 0.3 0 1.9 328 B L 3.7 3.6 3.8 4.4 50 8.47.0 50 3.8 0 2.6 329 B P 3.3 8.5 20 20 50 8.0 16.5 50 18.5 20 14.7 330 BA 0.5 2.0 2.8 0.5 2.4 3.9 1.2 4.0 0 2.0 2.1 331 B P 1.7 3.8 6.4 10.1 204.7 11.0 10.1 7.5 20 5.5 332 B I 1.7 2.9 1.3 1.7 14.8 7.0 13.9 1.7 3.1 01.7 333 B E 1.9 2.5 1.9 0 8.9 5.9 8.2 1.2 3.0 6.4 3.4 334 B K 2.9 3.93.7 2.6 20 8.3 12.1 1.5 2.6 5.3 3.7 335 B T 0 2.1 7.2 7.0 4.2 0.4 3.317.3 6.5 7.7 5.2 336 B I 0.5 1.6 2.1 0.7 20 5.0 6.1 0 1.3 5.3 2.1 337 BS 1.1 2.1 4.0 2.0 3.1 3.2 2.0 50 0 1.6 0.9 338 B K 0.6 2.3 3.0 3.0 9.45.3 10.6 2.2 1.1 0 3.2 339 B A 1.1 2.4 1.2 0.8 4.3 3.6 3.7 2.6 1.8 2.63.5 340 B K 0.9 2.0 1.4 0.8 3.0 3.4 2.9 2.1 0.8 2.5 2.3 Pos WT N P Q R ST V W Y 235 A L 1.0 0.3 1.4 1.8 0 1.9 3.6 6.6 3.3 236 A G 3.3 8.2 5.66.0 0.8 5.6 11.8 6.6 20 237 A G 20 50 50 50 20 20 50 50 50 238 A P 9.33.2 12.4 20 8.6 50 50 20 8.4 239 A S 1.8 9.1 1.3 2.5 0.3 5.7 10.7 2019.7 240 A V 2.0 1.1 9.5 13.1 2.5 0.5 0 20 20 241 A F 0.4 14.7 0.5 1.10.1 0 8.3 3.6 0.4 242 A L 5.5 0.9 7.9 17.1 3.8 2.3 0 20 17.5 243 A F 2.310.2 0.5 1.6 1.3 0.9 1.2 5.3 1.6 244 A P 2.8 2.0 0.9 1.7 0 19.3 20 7.612.2 245 A P 20 0 20 20 8.0 20 50 20 20 246 A K 0.2 0 1.2 1.5 1.7 1.41.2 5.4 3.0 247 A P 0 0.5 0.9 1.5 0.7 1.1 1.3 6.9 3.7 248 A K 1.2 3.61.5 2.3 0.7 0 2.5 5.6 2.7 249 A D 1.4 20 1.5 3.4 2.5 18.3 50 20 20 250 AT 3.2 50 8.7 9.3 1.8 1.3 1.9 20 50 251 A L 1.4 50 0 1.4 0.5 0.8 6.9 8.95.8 252 A M 0.3 17.4 0.1 1.1 0.1 0.2 4.6 4.2 3.3 253 A I 0.8 0.3 0 1.10.3 0.5 2.8 2.4 1.9 254 A S 0 20 0.3 1.2 0.3 0.8 0.7 3.8 1.9 255 A R 1.750 2.1 0 2.3 50 17.2 4.0 0.5 256 A T 0 0.4 0.1 0.2 0.4 0.9 1.2 5.6 2.7257 A P 20 0.1 13.1 20 2.9 16.0 20 50 50 258 A E 2.9 10.4 7.4 6.0 1.06.2 17.6 20 1.0 259 A V 4.1 50 9.2 20 5.2 2.1 0 20 20 260 A T 1.8 1.10.8 0.9 1.7 0.4 1.9 7.1 20 261 A C 20 50 20 20 3.6 20 20 20 20 262 A V0.6 50 3.8 5.2 3.4 3.0 1.7 20 20 263 A V 5.2 50 19.8 17.7 2.8 1.4 0 2020 264 A V 2.1 2.3 2.6 2.7 2.2 1.1 0.6 3.9 0.1 265 A D 5.5 50 10.2 8.67.9 20 50 20 5.7 266 A V 7.1 50 12.2 20 6.1 3.8 0 20 20 267 A S 4.7 02.3 3.1 3.0 20 20 50 50 268 A H 1.7 0 1.4 1.7 1.1 0.2 0.9 6.1 3.7 269 AE 0 50 0.6 0.8 0.2 0.6 0.7 4.0 1.0 270 A D 1.9 20 1.9 1.8 1.2 1.7 4.15.1 7.0 271 A P 7.3 5.9 5.9 5.9 1.6 4.1 15.2 20 20 272 A Q 0.3 50 1.11.6 0 1.0 3.5 4.0 3.4 273 A V 0 2.8 20 20 2.1 1.4 1.7 20 20 274 A K 0.920 0 0.1 0 0.4 0.7 3.3 2.3 275 A F 9.1 6.1 9.1 15.1 9.6 7.2 6.1 13.5 4.3276 A N 1.8 50 1.6 2.5 1.2 0 0.3 4.2 3.6 277 A W 6.6 3.5 5.5 15.4 6.96.1 14.1 0 20 278 A Y 0 50 1.9 2.2 2.6 9.9 20 15.8 1.4 279 A V 3.3 201.9 4.6 4.3 3.4 4.2 20 20 280 A D 3.8 50 0 3.7 0.6 6.8 12.7 11.9 11.4281 A G 50 50 50 50 50 50 50 50 50 282 A V 0.4 18.9 0.5 1.0 0 0.6 0.94.7 3.1 283 A E 0 0.4 0.6 1.5 0.4 0.3 1.2 4.1 0.9 284 A V 2.6 50 0.8 0.70.8 0.1 1.5 20 20 285 A H 0.7 2.2 0.2 0.8 0 1.1 4.7 4.9 4.0 286 A N 0.620 1.2 0.7 0.9 1.7 2.1 5.2 2.7 287 A A 3.6 50 2.6 2.3 1.0 1.9 12.5 9.110.4 288 A K 0.3 50 0.5 1.3 0 0.4 2.0 4.5 3.6 289 A T 2.1 8.2 1.2 2.0 00.4 12.0 3.9 3.2 290 A K 0 50 0.7 2.0 0.3 1.3 3.3 5.6 3.3 291 A P 1.20.7 0 2.9 2.2 0.9 1.3 2.6 0.9 292 A R 1.1 8.4 0.2 0.4 1.0 1.3 4.7 8.35.7 293 A E 7.2 5.5 15.1 14.5 3.5 20 50 14.5 17.1 294 A E 1.0 50 1.3 1.30.5 0 3.4 11.2 10.2 295 A Q 50 50 50 50 50 50 50 50 50 296 A Y 0.9 500.2 1.8 1.3 4.7 4.8 18.2 20 297 A N 9.3 50 4.4 4.4 1.5 1.6 15.5 20 20298 A S 3.3 50 1.7 2.1 0 2.2 7.3 15.6 20 299 A T 3.6 50 2.2 2.5 1.1 2.25.4 3.6 1.4 300 A Y 2.3 50 3.3 4.0 2.6 1.1 1.1 11.0 2.4 301 A R 2.5 502.6 2.3 2.9 1.8 0.9 9.8 1.8 302 A V 4.8 50 4.7 3.2 4.3 7.7 3.8 0 8.4 303A V 3.1 1.0 2.1 2.9 0.4 0.4 2.9 10.9 6.2 304 A S 16.6 50 20 16.6 2.214.2 17.9 20 20 305 A V 1.1 3.9 1.1 1.4 1.2 0.9 0 0.8 0.8 306 A L 6.0 505.9 9.9 6.2 5.3 11.4 9.6 10.3 307 A T 2.2 0 1.9 1.3 1.4 0.9 1.2 6.2 6.5308 A V 7.6 50 7.7 15.5 0 0.7 5.9 50 50 309 A L 0 1.6 0.7 1.3 1.0 0.60.5 5.0 2.1 310 A H 0 0.2 4.9 10 2.0 2.5 6.4 50 50 311 A Q 0.9 1.7 0.80.9 0 0.3 2.2 4.6 2.0 312 A N 7.1 50 2.7 3.9 4.1 3.2 11.9 20 20 313 A W5.4 50 4.8 12.9 6.0 3.8 6.6 0 2.6 314 A L 2.3 50 1.6 1.6 3.0 4.7 6.3 8.06.0 315 A D 0.6 50 0 0.7 0 0.9 2.4 6.2 3.7 316 A G 50 50 50 50 50 50 5050 50 317 A K 20 15.9 17.2 13.5 2.8 9.2 20 50 50 318 A E 1.7 20 1.4 2.62.2 1.3 0 6.1 9.5 319 A Y 3.2 50 3.1 5.6 3.4 3.6 20 20 0.2 320 A K 1.350 2.4 1.9 3.3 3.3 7.2 20 20 321 A C 20 50 19.6 20 1.5 8.7 18.3 20 20322 A K 2.7 50 2.1 0 2.3 1.6 0.9 14.5 2.8 323 A V 8.5 50 13.6 20 2.8 1.60 20 20 324 A S 0.9 50 0.8 2.9 2.7 1.9 2.1 3.8 2.5 325 A N 1.6 13.4 0.520 3.1 0.1 1.3 20 20 326 A K 1.2 0 0.6 1.4 1.0 1.9 2.6 5.6 3.6 327 A A5.3 20 1.3 4.1 0 5.2 13.7 20 20 328 A L 6.0 50 3.7 8.2 6.6 50 50 20 50329 A P 0 0.3 0.7 1.5 0.1 1.1 1.1 6.2 3.6 330 A A 0 20 0.6 1.2 0.2 0.61.9 7.0 3.4 331 A P 50 50 50 50 50 50 50 50 50 332 A I 3.9 20 0.8 2.42.3 2.6 4.4 20 5.9 333 A E 2.5 1.6 1.3 3.2 1.3 1.4 7.7 4.0 4.8 334 A K1.5 4.4 0.1 2.7 2.2 0.9 1.3 4.9 1.8 335 A T 1.2 0 2.3 2.8 1.4 1.4 7.35.1 4.5 336 A I 3.2 20 2.8 1.4 0.7 0.6 0 20 20 337 A S 5.0 50 11.4 12.74.5 2.3 50 19.3 10.6 338 A K 1.0 50 1.5 0.9 0.7 10.3 50 5.4 4.9 339 A A0 0.8 0.6 1.6 0.6 0.9 2.4 6.8 3.8 340 A K 1.0 1.9 0.9 1.3 0.5 0.8 1.74.9 2.4 232 B P 1.1 0 1.1 1.6 0.7 1.4 3.0 6.2 4.1 233 B E 0.6 2.7 0.41.6 0 1.2 6.9 5.5 2.6 234 B L 2.6 20 3.6 1.2 3.1 2.5 3.4 13.4 0.5 235 BL 1.9 17.3 0.6 1.4 0.8 0.7 5.2 7.8 5.3 236 B G 3.5 50 5.5 19.9 2.6 20 2020 14.1 237 B G 50 50 50 50 50 50 50 50 50 238 B P 8.1 1.3 5.8 20 4.94.4 1.3 20 20 239 B S 1.9 50 1.7 1.1 1.5 1.5 5.2 20 5.2 240 B V 7.6 011.6 20 1.2 1.9 0.8 20 20 241 B F 0.2 50 0.3 1.5 0.1 0.9 5.7 4.1 1.1 242B L 5.3 0 9.1 6.8 2.9 1.1 0.5 20 8.7 243 B F 0 50 1.6 2.7 0.1 1.8 3.94.3 1.0 244 B P 2.0 1.8 1.2 1.3 0 19.6 20 9.1 11.0 245 B P 20 0 20 206.0 20 50 20 20 246 B K 0.2 0.3 0.2 0.3 0.1 0 2.0 4.9 2.4 247 B P 0.3 00.8 1.5 0.3 0.7 9.5 6.6 3.4 248 B K 0 1.3 0.8 1.7 0.5 0.7 2.8 4.7 2.3249 B D 0.5 50 4.7 6.3 3.5 6.1 50 20 7.2 250 B T 9.2 50 3.4 4.9 1.3 2.33.1 20 20 251 B L 0.5 50 0.6 1.8 0.4 2.5 8.7 8.2 5.9 252 B M 1.0 50 1.01.6 1.5 1.3 0.8 5.1 1.6 253 B I 1.1 1.0 0 1.5 1.2 1.4 3.4 4.4 3.6 254 BS 0.3 6.2 0.5 1.7 0 0.1 1.1 5.5 3.7 255 B R 0.8 50 1.4 1.1 1.5 20 20 3.70.8 256 B T 0 1.2 0.5 0.1 0.8 1.2 1.2 5.5 2.4 257 B P 20 0 20 20 4.8 2020 50 50 258 B E 1.1 50 1.3 2.5 2.2 1.1 1.0 19.1 3.0 259 B V 5.6 50 6.220 4.5 2.5 0 20 20 260 B T 3.9 0.2 2.3 2.6 0.4 0.1 2.7 20 20 261 B C 2020 20 20 2.0 16.6 20 20 20 262 B V 2.7 50 3.2 4.8 2.9 1.9 0 14.7 9.1 263B V 5.4 50 13.0 20 3.6 2.1 0 20 20 264 B V 3.6 10.1 3.0 2.2 2.6 2.2 1.012.7 20 265 B D 1.8 50 4.5 5.3 4.5 6.0 9.2 12.2 5.6 266 B V 5.7 50 18.320 5.9 4.7 0 50 50 267 B S 3.2 0.5 1.5 0.8 3.3 11.6 50 6.3 50 268 B H2.6 3.4 2.1 1.8 2.5 3.8 2.7 7.8 5.5 269 B E 0 12.8 0.5 0.7 0.3 0.7 0.65.1 2.7 270 B D 1.2 5.9 6.3 2.1 0.3 1.9 5.4 16.3 5.6 271 B P 3.3 7.4 2.70 1.5 2.2 5.2 4.8 4.4 272 B Q 0.6 4.9 0 1.4 0.2 0.6 1.4 3.9 3.2 273 B V7.4 50 10.6 20 2.0 0 4.8 20 20 274 B K 0.3 15.6 0.1 0 0 0.2 1.6 2.2 1.9275 B F 7.0 4.1 9.7 12.3 6.9 4.5 3.3 10.3 5.0 276 B N 3.8 50 6.4 9.2 2.820 20 20 20 277 B W 10.8 6.8 9.3 11.9 9.7 8.0 14.4 0 15.9 278 B Y 12.611.0 4.4 2.0 0.8 2.5 19.8 20 4.2 279 B V 3.4 20 1.4 4.0 4.2 2.4 1.2 2020 280 B D 1.6 20 1.4 3.1 0 2.7 5.5 8.1 7.3 281 B G 3.4 50 4.0 5.3 3.63.2 6.4 10.3 7.6 282 B V 0.2 50 0 0.7 0 0.4 0.7 6.1 2.8 283 B E 2.5 01.5 1.6 1.0 1.5 3.9 7.9 6.7 284 B V 2.4 50 1.5 3.3 0 1.5 1.8 20 20 285 BH 0 1.6 0.9 0.8 0.5 0.4 2.0 5.8 2.4 286 B N 0 50 0.4 1.6 1.1 0.5 2.9 4.93.0 287 B A 0.3 12.3 8.1 9.1 4.1 3.3 7.1 3.4 0.8 288 B K 0.9 15.4 0.61.1 0.2 0.9 3.8 5.9 2.7 289 B T 1.5 1.8 1.1 1.7 0 0.4 2.3 3.4 2.5 290 BK 0.2 50 0.7 1.5 0 0.6 2.9 5.0 2.7 291 B P 1.1 0.6 0.1 1.1 1.1 0.9 1.53.2 2.6 292 B R 2.2 16.6 1.5 1.8 0.1 0 3.2 7.6 5.2 293 B E 2.2 1.3 2.22.5 0 1.2 7.8 7.0 6.9 294 B E 4.1 0 3.3 5.0 2.1 2.9 5.0 6.7 11.9 295 B Q1.0 0.4 0.4 1.1 0 3.9 6.6 6.1 3.5 296 B Y 0 20 0.6 1.9 1.2 1.4 1.3 6.44.0 297 B N 7.3 20 4.4 4.2 3.6 4.1 7.9 18.0 15.0 298 B S 0.4 50 1.2 1.00.9 2.2 3.3 5.5 2.0 299 B T 4.8 50 9.8 17.9 0.3 1.3 5.8 20 20 300 B Y6.5 50 4.0 3.8 4.3 3.6 9.1 20 6.4 301 B R 1.6 14.1 0.6 0.4 1.8 1.1 017.9 20 302 B V 4.7 9.6 4.1 0.6 4.3 2.0 0 20 0.2 303 B V 2.0 8.6 2.0 4.70.6 0.5 1.3 20 20 304 B S 6.3 50 20 20 0 2.7 3.8 20 20 305 B V 1.8 500.6 2.0 0.6 0 0.7 20 20 306 B L 5.7 13.4 4.4 14.1 5.5 4.3 6.0 20 12.1307 B T 1.7 0 1.7 1.9 1.5 1.4 2.0 4.4 4.3 308 B V 12.4 50 20 20 1.2 3.64.3 20 20 309 B L 0.2 0 1.6 2.3 1.8 14.3 20 5.1 3.3 310 B H 0 2.3 4.67.0 1.8 1.6 3.8 20 20 311 B Q 0.6 3.2 0.4 0.8 0.2 1.6 18.8 4.6 2.0 312 BN 0.9 50 1.3 5.7 0.1 5.6 3.8 8.0 7.8 313 B W 8.0 50 6.5 8.9 6.6 17.2 202.1 0.9 314 B L 1.9 50 0.8 1.0 1.7 0.9 3.1 3.7 11.3 315 B D 1.8 50 1.02.2 0.2 0 4.5 8.5 6.8 316 B G 50 50 50 50 50 50 50 50 50 317 B K 1.3 504.2 0.9 0.4 13.8 10.1 20 1.7 318 B E 2.3 3.8 1.0 1.6 0.4 0 1.0 3.8 7.7319 B Y 7.2 50 9.0 13.7 7.2 5.8 3.9 20 1.7 320 B K 6.6 50 2.9 2.4 4.03.3 2.0 20 20 321 B C 20 19.7 20 20 3.1 11.2 20 20 20 322 B K 2.2 50 0.90.3 3.3 1.6 0 20 20 323 B V 10.5 50 8.7 20 5.6 4.6 0 20 20 325 B N 0 506.3 20 4.6 8.8 17.8 20 20 326 B K 0.1 4.4 0 1.1 0.1 3.2 2.1 5.2 0.7 327B A 1.9 20 3.0 2.3 3.3 20 20 20 20 328 B L 4.0 50 4.2 8.7 4.8 2.9 12.350 50 329 B P 20 0 20 20 1.4 17.1 16.4 50 50 330 B A 0.8 20 0 0.5 0.80.2 4.6 8.2 2.6 331 B P 5.0 0 7.6 7.4 2.6 20 10.1 17.6 20 332 B I 1.7 501.8 5.3 2.0 1.9 3.4 20 20 333 B E 2.0 3.1 1.1 2.3 1.8 1.6 1.6 8.9 9.3334 B K 4.3 50 1.9 0 3.4 1.8 1.4 9.9 20 335 B T 5.5 3.5 7.0 5.7 0.2 5.511.5 5.2 3.1 336 B I 1.8 20 0.6 3.1 1.1 0.8 0.7 19.4 20 337 B S 1.9 15.81.1 2.2 1.4 50 50 5.5 3.9 338 B K 1.5 16.2 2.7 2.7 1.1 2.8 3.5 8.1 11.0339 B A 0 2.3 0.5 1.3 0.2 0.8 2.0 6.7 3.8 340 B K 0.2 1.0 0.1 1.2 0 0.52.0 5.5 3.2

SPA™ technology; 1IIS template structure; −carbohydrate, no floatedpositions TABLE 56 Pos WT A C D E F G H I K L M 239 A S 0.2 4.6 2.7 0 204.6 14.5 11.0 1.9 0.3 2.0 240 A V 1.5 2.4 2.4 6.9 20 7.4 20 5.1 9.9 5.95.5 263 A V 2.3 2.8 6.3 16.5 20 8.8 20 9.6 7.3 7.3 15.3 264 A V 1.8 3.12.6 1.8 0 6.3 1.9 0.6 2.4 0.8 2.7 266 A V 4.9 5.2 6.9 12.3 20 11.1 200.8 11.9 20 8.5 296 A Y 3.4 2.7 1.1 0 50 0.7 50 5.0 3.6 3.5 4.2 299 A T0.7 3.2 9.9 10.4 20 6.2 20 10.7 6.7 20 4.1 325 A N 2.5 3.5 7.7 2.5 208.0 20 0 6.1 20 7.8 328 A L 6.1 6.3 7.1 4.2 50 8.8 20 50 4.6 0 7.2 330 AA 0.9 1.8 1.2 0 2.5 4.0 2.9 1.7 1.2 1.6 2.8 332 A I 1.9 3.8 4.6 1.3 5.17.1 1.8 3.4 0.2 0 2.6 239 B S 1.0 2.4 3.5 2.0 6.7 5.6 2.9 3.1 0.3 0 1.9240 B V 0.3 2.4 6.9 11.7 20 6.6 20 8.3 12.3 20 14.2 263 B V 2.4 3.9 4.512.5 20 9.3 20 15.8 17.1 2.1 20 264 B V 2.2 3.2 4.8 2.7 7.4 6.9 6.0 01.9 1.9 3.8 266 B V 5.4 5.5 7.5 13.2 20 12.1 20 2.6 20 20 20 296 B Y 1.52.7 1.3 1.2 4.0 4.1 3.6 1.1 1.9 2.6 3.5 299 B T 0 2.2 7.5 10.2 20 4.8 207.7 5.8 20 10.3 325 B N 3.4 5.1 8.6 5.0 20 8.2 20 16.7 20 20 20 328 B L3.6 3.5 3.8 3.9 50 8.3 7.0 50 2.9 0 1.9 330 B A 0.7 2.1 2.9 0.7 2.7 4.01.4 4.8 0 2.2 2.3 332 B I 1.8 2.9 1.2 1.8 13.5 7.0 9.9 1.7 3.2 0 1.7 PosWT N P Q R S T V W Y 239 A S 1.9 8.1 1.4 2.6 0.4 5.7 11.6 20 20 240 A V2.4 1.1 12.3 13.1 2.6 0.5 0 20 20 263 A V 4.8 50 16.4 17.4 2.8 1.4 0 2020 264 A V 2.1 1.6 2.3 2.7 2.3 1.1 0.5 3.5 0 266 A V 6.6 50 12.5 20 6.13.7 0 20 20 296 A Y 0.9 50 0.9 2.9 2.2 5.3 5.5 16.1 18.4 299 A T 12.9 505.9 11.8 0 2.5 8.2 13.3 20 325 A N 1.2 12.8 0.8 20 2.7 0 1.0 20 20 328 AL 6.1 50 4.0 8.3 6.7 50 50 20 50 330 A A 0 20 0.4 1.0 0.2 0.5 1.7 6.22.9 332 A I 3.8 20 0.6 2.4 2.3 2.5 4.2 20 5.6 239 B S 2.1 50 1.5 1.8 1.41.4 5.2 20 4.2 240 B V 7.4 0 13.4 20 1.3 1.9 0.9 20 20 263 B V 5.3 5013.8 20 3.9 2.2 0 20 20 264 B V 3.7 9.9 3.1 2.2 2.7 2.4 0.9 14.7 18.2266 B V 5.4 50 16.1 20 6.0 4.7 0 50 50 296 B Y 0 20 0.7 1.8 1.1 1.4 1.36.5 4.2 299 B T 5.1 50 10.2 18.4 0.3 1.1 5.4 20 20 325 B N 0 19.7 6.3 204.6 8.6 18.2 20 20 328 B L 3.8 50 3.4 8.4 4.7 2.9 12.5 50 50 330 B A 0.820 0.2 0.8 1.1 0.2 4.7 7.8 3.2 332 B I 1.9 50 1.2 5.4 2.0 2.0 3.3 20 20

SPA™ technology; D129G 1IIS template structure; +carbohydrate TABLE 57Pos WT A C D E F G H I K L M 239 A S 1.2 3.5 1.7 0 20 5.8 11.0 6.6 2.93.9 3.9 240 A V 1.2 2.4 6.0 14.0 20 7.1 20 6.7 9.4 10.1 7.5 263 A V 00.4 1.0 8.7 20 6.9 4.4 11.7 4.9 16.0 19.2 264 A V 2.9 3.7 6.3 2.8 11.67.6 13.2 0 3.2 3.4 4.1 266 A V 4.8 5.9 6.8 9.5 50 10.3 20 3.5 12.7 12.212.7 296 A Y 0.8 2.0 1.5 0.1 0.2 3.4 1.5 6.6 1.7 0.6 1.8 299 A T 1.9 3.77.5 0 20 7.9 14.2 2.9 0.8 3.4 4.4 325 A N 1.0 1.4 3.1 2.8 20 7.4 20 8.57.7 10.4 6.1 328 A L 2.5 5.3 4.0 1.9 50 7.5 20 20 1.6 0.2 0 330 A A 0.92.1 1.8 1.2 2.4 2.7 3.1 3.1 1.4 2.1 3.5 332 A I 2.9 3.7 3.9 0.9 6.1 7.82.5 0 2.7 0.8 2.8 239 B S 1.9 3.1 3.0 1.9 1.5 6.2 2.3 14.1 1.8 1.4 2.9240 B V 0.5 1.7 5.0 13.3 20 6.6 20 1.2 12.4 12.1 8.8 263 B V 2.9 3.2 6.418.2 10.1 9.2 6.9 12.8 6.0 20 10.3 264 B V 2.9 3.6 4.4 3.0 8.8 7.1 6.2 02.3 1.9 4.5 266 B V 4.4 4.6 2.6 6.6 20 10.7 20 0 4.9 1.7 8.5 296 B Y 07.1 6.7 7.2 20 0.1 18.6 50 7.0 2.7 6.6 299 B T 0 3.2 10.4 6.0 20 5.5 2015.9 3.2 5.9 4.4 325 B N 1.4 2.5 5.0 0 20 7.0 20 20 1.0 2.2 1.0 328 B L0.4 1.3 5.6 0 50 4.5 50 50 1.9 2.4 2.4 330 B A 0.6 1.4 2.5 0.9 3.1 2.51.2 20 0 2.4 2.1 332 B I 4.3 5.3 5.7 0 11.4 9.3 43 2.5 5.8 2.0 4.0 PosWT N P Q R S T V W Y 239 A S 2.7 8.5 1.3 2.7 0.6 3.5 5.4 20 20 240 A V4.4 1.8 14.8 20 2.0 0.4 0 20 20 263 A V 0.8 50 11.7 20 1.4 0.1 1.0 20 20264 A V 4.2 7.1 2.9 3.4 3.1 1.9 0.8 12.8 16.3 266 A V 4.1 50 11.9 11.95.2 2.9 0 50 50 296 A Y 1.2 2.6 0 1.6 0.2 2.5 5.6 3.8 0 299 A T 2.3 501.9 3.0 3.5 4.1 3.3 20 20 325 A N 2.8 15.4 5.4 20 0 0.1 3.8 20 20 328 AL 2.9 50 0.4 4.8 3.2 2.9 7.0 50 50 330 A A 0.5 20 0.8 1.0 0 0.5 2.9 5.22.9 332 A I 3.5 50 0.7 3.7 2.9 2.5 1.0 8.1 6.9 239 B S 1.8 0 1.9 3.2 1.92.3 7.7 6.6 15.8 240 B V 4.6 6.3 20 20 1.0 0.2 0 20 20 263 B V 5.7 5017.5 20 3.2 2.2 0 20 20 264 B V 3.4 1.7 3.2 3.5 3.5 2.0 0.9 12.0 16.4266 B V 5.6 50 6.0 12.4 5.3 4.6 1.5 20 50 296 B Y 6.8 50 7.2 9.3 2.3 5050 20 14.1 299 B T 6.4 50 5.7 9.4 1.2 1.4 13.7 20 20 325 B N 0.3 1.9 1.120 2.6 5.1 20 20 20 328 B L 8.3 50 0.8 16.4 1.0 1.2 50 50 50 330 B A 0.320 0.4 0.6 0 4.0 20 13.5 3.4 332 B I 6.5 17.9 3.7 5.9 4.6 4.2 3.7 2011.6

SPA™ technology; D129G 1IIX template structure; +carbohydrate TABLE 58Pos WT A C D E F G H I K L M 239 A S 1.2 2.3 2.2 1.8 7.9 5.5 7.6 0.5 0.21.8 2.6 240 A V 0.7 2.9 6.8 4.3 20 6.5 20 0 10.7 20 3.1 263 A V 1.7 2.94.6 18.8 20 8.4 5.8 15.1 2.3 14.5 2.1 264 A V 2.7 3.3 3.6 1.5 13.9 6.75.9 0 2.3 4.9 3.7 266 A V 3.5 3.5 5.7 12.4 20 10 20 5.7 6.3 7.8 7.4 296A Y 2.6 50 50 50 50 0 50 50 18.5 18.0 50 299 A T 0.2 0.7 6.6 1.2 20 5.69.6 1.6 0.8 1.5 1.8 325 A N 3.1 3.6 7.3 2.4 20 7.7 20 20 20 10 13.1 328A L 0.6 0 1.5 5.4 50 1.6 50 50 3.1 4.2 9.6 330 A A 1.9 2.5 4.1 2.8 4.54.1 3.0 3.2 1.0 2.7 3.5 332 A I 2.3 3.5 2.2 0.8 20 6.8 9.6 0 3.4 0.2 2.6239 B S 1.4 3.6 2.5 1.4 16.8 5.8 6.2 5.0 2.5 1.4 2.0 240 B V 0 2.6 12.818.6 20 5.7 20 12.7 10.4 20 8.5 263 B V 1.1 2.4 3.6 20 20 7.8 17.7 11.84.5 20 6.3 264 B V 3.3 4.0 5.0 2.9 14.2 7.5 4.8 0 2.6 3.6 4.6 266 B V2.9 3.3 4.9 11.3 50 9.5 20 20 20 7.9 15.0 296 B Y 2.8 50 50 50 50 0 5050 17.7 18.7 50 299 B T 0 3.8 12.6 9.2 20 5.9 20 7.3 4.8 3.2 4.3 325 B N0.3 2.0 5.5 2.2 50 6.1 20 0 10.5 15.5 14.6 328 B L 5.4 5.7 7.3 4.4 509.8 20 50 2.5 0 5.1 330 B A 0.6 1.4 3.2 1.3 3.9 3.2 2.7 4.0 1.3 3.7 3.1332 B I 1.9 3.1 2.7 1.7 5.2 6.9 3.1 0.4 1.3 0 1.9 Pos WT N P Q R S T V WY 239 A S 1.4 0.9 1.3 1.9 1.5 0.8 0 8.6 9.6 240 A V 9.1 2.1 7.7 20 1.41.1 2.4 20 20 263 A V 3.2 50 20 15.0 3.6 1.2 0 20 20 264 A V 3.2 1.9 2.53.0 3.0 2.5 0.7 19.9 19.0 266 A V 5.2 50 16.6 20 4.2 1.7 0 20 50 296 A Y50 50 50 50 50 50 50 50 13.6 299 A T 4.8 50 1.0 9.2 0 0 1.6 20 20 325 AN 3.6 50 0 20 4.0 9.7 20 20 20 328 A L 1.4 50 6.9 9.6 0.6 0.1 50 50 50330 A A 2.1 20 2.4 2.6 1.3 0 3.9 7.6 5.3 332 A I 2.8 14.5 3.3 4.6 2.61.3 0.9 10.5 20 239 B S 3.8 0.3 0.5 2.4 0 1.6 5.3 20 19.5 240 B V 15.13.1 20 20 1.0 0.2 2.4 20 20 263 B V 3.3 50 20 20 3.2 1.2 0 20 20 264 B V3.5 1.7 3.1 4.1 3.9 2.9 1.3 6.9 20 266 B V 4.5 50 4.9 20 1.9 0 3.6 50 50296 B Y 50 50 50 50 50 50 50 50 11.3 299 B T 8.0 50 12.3 8.8 0.2 2.1 4.420 20 325 B N 1.3 10 2.4 20 2.3 2.0 1.0 20 50 328 B L 5.9 50 2.8 7.4 6.16.4 50 50 50 330 B A 0.7 20 0.6 1.3 0 0.4 4.2 8.2 3.6 332 B I 2.6 7.71.3 2.2 2.3 1.6 2.0 10.4 5.6

SPA™ technology; D129G 1E4K template structure; +carbohydrate TABLE 59Pos WT A C D E F G H I K L M 239 A S 1.4 2.6 3.1 1.0 20 5.7 4.8 3.4 2.01.2 2.6 240 A V 2.9 3.5 3.7 4.6 20 8.2 10.8 0 9.1 3.2 5.4 263 A V 3.64.9 6.2 8.7 20 9.9 20 3.7 4.2 0.5 6.7 264 A V 1.8 2.8 3.3 2.0 2.9 6.23.1 0 2.4 0.8 3.0 266 A V 4.4 5.2 4.9 7.1 20 10.6 20 1.0 12.1 4.8 9.1296 A Y 1.2 2.9 0.7 1.4 3.1 3.9 2.7 2.4 2.3 1.9 2.2 299 A T 0 2.6 6.011.5 20 5.3 20 20 6.0 20 4.4 325 A N 5.2 7.0 6.6 6.9 50 11.3 20 1.3 14.313.5 13.9 328 A L 4.8 5.5 7.0 3.2 20 10.5 20 50 5.1 0 8.5 330 A A 0.91.8 1.1 0.9 3.5 4.0 3.0 2.3 1.2 1.6 2.8 332 A I 5.3 6.4 6.7 4.8 8.2 9.95.2 3.1 0 3.6 5.2 239 B S 0.7 2.3 2.6 2.0 5.3 5.1 3.3 1.7 0 0 2.0 240 BV 2.3 3.0 4.1 7.3 20 8.1 20 5.1 20 11.8 10.9 263 B V 3.2 4.3 7.3 8.3 209.6 20 13.3 8.5 0.6 20 264 B V 2.1 3.2 3.7 2.7 17.8 6.6 11.5 0 2.0 0.83.5 266 B V 5.0 5.0 5.2 16.3 20 11.2 20 2.3 20 14.3 17.3 296 B Y 0.9 2.31.0 0.5 2.7 3.7 2.5 1.2 1.3 2.1 3.0 299 B T 1.1 2.2 7.6 5.4 20 6.4 12.81.8 3.9 17.5 6.9 325 B N 10.1 11.5 13.1 11.2 20 15.7 20 8.6 14.3 17.1 20328 B L 2.9 4.1 4.8 3.5 50 8.5 1.7 9.6 1.5 0 1.5 330 B A 0.1 2.0 1.4 1.81.6 4.0 3.0 2.0 0.5 0.5 2.6 332 B I 3.4 4.4 3.5 3.1 6.1 8.2 4.1 0 3.31.3 3.3 Pos WT N P Q R S T V W Y 239 A S 1.6 4.8 0 2.1 1.3 2.1 3.3 13.819.6 240 A V 3.1 4.8 5.5 17.5 4.0 1.8 1.2 20 20 263 A V 6.1 50 9.5 205.1 3.6 0 20 20 264 A V 2.4 6.1 1.4 2.8 2.4 1.9 0.8 10.2 2.2 266 A V 4.650 7.9 12.6 5.8 3.5 0 20 20 296 A Y 0 1.6 1.4 3.0 0.9 1.0 3.5 6.0 2.6299 A T 3.0 50 14.1 13.2 0.9 3.8 15.1 15.0 20 325 A N 0 5.0 6.0 20 6.04.6 3.2 20 50 328 A L 5.5 50 3.5 8.2 5.5 13.4 50 20 50 330 A A 0 14.50.9 1.1 0.1 0.4 2.0 6.4 3.2 332 A I 6.8 20 3.5 4.6 5.5 4.8 4.0 11.2 7.1239 B S 0.8 15.5 0.9 0.8 0.7 0.7 3.3 8.2 6.0 240 B V 3.8 2.0 17.0 20 3.61.3 0 20 20 263 B V 6.0 50 8.5 20 4.6 4.0 0 20 20 264 B V 3.0 7.8 2.01.5 2.5 1.3 1.0 13.9 20 266 B V 2.5 50 11.6 20 5.4 3.9 0 20 20 296 B Y 07.0 0.4 1.1 0.3 0.8 1.8 6.0 2.4 299 B T 3.9 20 4.6 10.3 0.8 0 1.9 20 20325 B N 0 16.1 10.6 20 11.1 10.9 10.5 20 20 328 B L 3.5 50 3.3 2.0 3.31.9 5.2 50 50 330 B A 0 20 0.7 2.0 0.3 0.6 2.1 4.4 2.4 332 B I 4.0 15.70.8 2.1 3.9 2.7 1.1 20 6.1

SPA™ technology; Fc/FcγRIIb model template structure; −carbohydrateTABLE 60 Pos WT A C D E F G H I K L M 237 A G 1.0 3.0 5.9 1.8 5.0 4.73.1 1.4 1.4 4.2 3.7 238 A P 3.3 5.2 9.7 4.6 20 9.2 20 0.4 19.6 5.0 8.2239 A S 0.6 1.8 3.3 1.3 1.8 4.7 0.5 2.1 0 0.8 2.6 240 A V 1.3 2.3 2.78.2 20 7.0 20 6.1 8.7 0 9.2 241 A F 0 2.1 2.4 0.5 1.0 4.0 2.6 7.5 1.70.9 2.8 242 A L 3.7 4.7 6.6 6.7 20 9.2 20 13.0 13.8 0 7.0 243 A F 0.82.0 1.0 0.2 0.3 4.9 1.5 4.0 1.5 0.8 2.2 244 A P 2.8 3.7 5.5 3.2 6.4 2.93.6 0 0.1 4.0 3.7 245 A P 2.5 20 20 20 20 7.7 20 50 20 20 20 246 A K 2.13.5 2.8 2.5 4.0 6.5 4.5 3.4 2.7 1.9 4.1 247 A P 2.4 5.0 6.6 7.8 50 7.520 10 9.6 50 6.5 248 A K 2.0 4.0 4.8 4.2 20 6.9 9.6 3.3 0 2.7 3.0 249 AD 3.3 4.2 3.3 0.9 4.8 9.3 4.0 50 3.1 0 3.2 250 A T 0.1 1.5 3.0 8.0 206.7 20 2.5 5.4 9.5 5.7 251 A L 3.1 4.6 6.3 6.1 2.3 9.5 1.5 7.2 3.1 1.35.4 252 A M 3.0 3.5 5.3 2.8 20 9.4 20 4.4 0.6 5.0 0 253 A I 0.9 1.8 3.93.3 4.0 5.5 6.2 1.3 0 0.9 1.8 254 A S 0 2.4 5.5 3.4 8.1 3.9 8.5 4.2 3.31.7 5.0 255 A R 2.2 3.8 5.4 3.9 7.1 7.3 1.6 15.8 0 0.5 1.5 256 A T 1.12.3 1.7 1.5 3.2 3.9 2.5 3.1 1.1 2.0 2.7 257 A P 2.1 9.1 20 16.1 50 8.550 20 20 20 20 258 A E 0 1.6 2.3 0.6 0.9 4.2 2.1 9.4 2.4 3.5 1.9 259 A V4.0 4.3 7.0 11.4 20 10.4 20 3.2 14.0 14.9 10.6 260 A T 2.6 2.8 3.2 0 207.3 5.6 1.7 3.1 5.5 3.5 261 A C 0 18.6 20 20 20 3.8 20 20 20 20 20 262 AV 1.8 1.5 1.3 11.1 20 7.1 20 5.8 14.6 20 20 263 A V 4.5 5.1 6.5 14.1 2010.8 20 8.0 6.1 3.2 10.9 264 A V 2.6 3.2 3.7 2.0 14.0 7.0 9.0 0 2.6 2.53.8 265 A D 1.4 2.8 2.7 1.6 1.8 2.5 2.2 18.5 0 0.1 2.5 266 A V 4.8 5.15.0 14.2 20 11.5 20 0.2 20 9.8 20 267 A S 2.6 4.9 5.7 3.3 1.6 7.2 2.90.5 1.0 2.0 3.7 268 A H 1.5 1.9 2.6 2.6 4.4 4.8 2.7 1.7 0 2.8 1.9 269 AE 0.3 2.0 0.8 0.6 2.8 3.6 2.7 1.5 1.6 1.4 2.9 270 A N 0 1.4 1.9 3.8 0.73.9 2.2 6.8 2.4 3.5 2.7 271 A P 0.9 2.2 5.7 6.5 3.0 5.5 2.9 12.8 5.415.7 5.6 272 A E 0.7 1.8 0.3 0.4 3.0 3.6 2.6 1.7 1.4 2.0 2.9 273 A V 3.14.0 2.1 15.5 20 8.9 20 0.5 10.6 7.1 12.4 274 A K 0.6 2.1 1.5 0.9 2.2 4.32.6 1.6 1.3 1.2 2.9 275 A F 7.1 8.6 10.1 8.0 0 12.2 4.2 7.7 4.7 5.5 6.8276 A N 0.5 1.8 1.3 0 20 5.4 20 12.6 2.6 3.2 3.0 277 A W 9.3 11.1 12.310.7 4.1 15.0 8.6 10.2 7.3 7.8 7.9 278 A Y 0.1 1.9 6.1 0 2.7 5.5 4.516.4 1.3 16.3 1.5 279 A V 3.2 4.4 5.1 3.1 20 8.2 19.7 0.3 0 1.9 3.1 280A D 3.6 3.5 0 2.7 12.5 4.0 9.9 17.6 3.8 2.8 3.9 281 A G 50 50 50 50 50 050 50 50 50 50 282 A V 0.5 1.8 1.8 0.8 2.2 4.0 2.2 0.7 0.9 1.3 3.0 283 AE 0.9 1.2 4.9 0 7.9 4.4 4.2 2.2 1.3 4.7 1.3 284 A V 2.1 2.6 4.4 2.6 15.06.7 4.4 2.0 1.8 6.3 2.6 285 A H 1.0 2.3 1.9 1.3 2.8 2.8 2.6 2.0 1.6 2.22.8 286 A N 0.9 1.7 0.7 0.1 3.3 3.4 3.2 0 0.9 2.1 2.3 287 A A 2.4 4.35.1 8.4 0 8.4 3.1 11.2 6.6 17.4 4.4 288 A K 0.7 1.9 2.0 0.9 2.7 3.9 2.61.0 1.3 1.9 2.2 289 A T 1.0 1.7 2.2 0.5 6.6 4.7 2.3 0.5 0.3 0.5 1.6 290A K 1.1 2.6 2.6 1.6 3.8 5.6 5.4 3.1 0.8 0.4 2.2 291 A P 1.0 2.4 1.8 1.74.5 3.6 3.4 2.6 1.6 2.6 3.5 292 A R 1.5 3.2 3.2 1.9 1.7 5.2 3.0 1.8 1.00 1.6 293 A E 0.6 2.1 1.5 0.4 8.0 4.3 5.5 3.2 1.4 0.8 2.2 294 A E 2.32.5 0.5 0.5 5.0 5.8 3.5 4.1 1.5 2.0 2.7 295 A Q 3.1 3.2 3.8 2.8 17.6 7.812.8 9.7 1.3 0 2.3 296 A Y 3.3 3.5 3.6 2.3 4.7 0 3.5 50 2.6 3.6 4.7 297A N 1.4 2.3 3.2 1.4 4.4 4.3 3.0 0.6 0.7 2.2 2.4 298 A S 6.5 7.1 6.9 6.911.0 0 8.6 50 6.6 7.2 8.5 299 A T 0.3 2.2 3.0 20 0.5 4.4 0.5 5.1 1.516.2 3.3 300 A Y 4.1 4.8 5.1 5.2 0 9.8 2.2 11.9 3.3 2.6 3.7 301 A R 1.62.4 1.1 0.6 20 6.5 20 8.6 0 3.5 1.7 302 A V 3.2 4.0 5.0 4.0 20 9.0 5.60.3 1.6 2.3 2.7 303 A V 0.9 1.0 2.0 0 20 5.9 20 30 4.4 12.4 4.6 304 A S1.1 2.3 4.1 9.2 20 7.8 20 7.6 6.9 20 9.3 305 A V 1.6 2.1 1.8 4.1 20 6.320 1.0 3.9 2.9 3.0 306 A L 5.1 6.8 6.7 7.0 1.7 11.5 4.9 3.8 3.9 0 6.3307 A T 1.5 3.0 2.6 1.6 1.1 5.5 3.0 0.2 1.7 1.0 3.0 308 A V 4.0 4.5 12.87.2 20 10.2 20 0 17.1 5.0 20 309 A L 1.3 2.8 1.8 1.7 3.2 5.5 3.3 0.8 1.51.0 3.2 310 A H 1.6 2.2 2.1 4.0 18.6 6.3 4.2 3.5 3.2 6.7 3.4 311 A Q 0.21.6 1.4 0.7 1.9 3.9 1.6 0.4 0.7 0.7 1.9 312 A D 0 1.6 0.8 0.9 20 5.311.3 8.9 2.4 2.3 2.5 313 A W 4.0 5.5 7.0 5.1 0 10 5.1 11.0 2.7 4.2 2.7314 A L 2.8 4.3 5.8 3.7 20 7.6 7.3 4.2 1.8 0 2.9 315 A N 0 5.1 3.1 3.411.1 2.6 11.3 16.1 3.4 3.4 4.8 316 A G 11.4 10 11.1 8.9 16.6 0 12.7 508.5 8.8 9.4 317 A K 3.1 4.8 8.1 5.6 6.5 8.4 2.5 7.3 0 5.5 3.5 318 A E1.5 2.5 2.8 1.7 20 5.8 9.6 2.0 1.9 5.9 3.4 319 A Y 7.0 7.9 9.4 9.9 012.8 4.6 3.2 6.0 6.6 7.1 320 A K 1.8 2.9 6.7 1.8 20 7.1 20 1.1 0 8.1 2.2321 A C 0 3.9 20 20 20 6.1 20 20 20 20 20 322 A K 2.4 3.3 6.1 3.0 20 8.020 1.1 0.4 16.9 2.5 323 A V 3.5 4.3 8.0 9.0 20 9.9 20 4.7 10.2 20 5.7324 A S 0.4 2.0 0.9 0 0.2 5.0 2.5 1.3 1.3 0 2.4 325 A N 4.9 5.9 6.3 6.020 10.4 20 1.7 14.0 11.3 20 326 A K 1.6 3.6 2.4 2.4 3.8 4.9 3.0 10.6 1.42.9 4.2 327 A A 2.7 3.8 5.5 2.8 11.6 7.1 9.5 3.2 1.2 3.7 4.3 328 A L 2.53.7 4.6 1.7 50 7.4 20 5.7 1.8 0 10.4 329 A P 0.8 2.2 1.2 1.2 4.0 3.3 3.32.9 1.6 2.5 3.5 330 A A 0.3 1.6 2.0 1.3 3.4 3.2 2.3 2.4 1.3 2.5 3.0 331A P 1.5 3.5 8.0 10.4 6.6 6.3 5.0 9.3 8.1 12.6 6.4 332 A I 2.4 3.5 2.62.2 7.9 7.2 4.0 1.3 2.3 0.7 2.9 333 A E 2.0 2.7 2.6 0 8.0 6.3 7.6 3.13.5 5.9 3.0 334 A K 2.1 3.4 3.5 2.0 7.1 7.1 3.5 2.0 2.2 3.0 3.0 335 A T0.5 1.0 2.1 0.3 4.6 4.6 4.1 0.6 0.2 4.0 2.1 336 A I 0.4 1.4 2.2 0.9 13.94.8 4.7 0 1.3 4.3 1.6 337 A S 0.3 0.8 4.0 13.5 20 3.1 20 50 9.9 9.3 5.6338 A K 4.7 8.3 7.1 7.6 20 9.8 20 5.5 0 2.3 5.9 339 A A 1.4 2.7 3.1 2.22.3 5.5 3.3 0 0.5 0.2 2.7 340 A K 1.2 3.0 2.0 1.5 1.9 3.9 2.5 2.6 0.22.2 2.1 237 B G 0.6 2.2 1.7 0.5 5.0 4.2 2.5 0.3 0.3 4.1 1.7 238 B P 3.05.3 8.9 5.4 20 9.1 20 1.1 14.4 3.6 11.6 239 B S 0.7 2.0 2.9 1.1 2.2 4.80.5 2.9 0 0.8 2.5 240 B V 2.0 3.0 3.4 5.3 20 7.7 20 6.4 11.3 0 10.2 241B F 0.3 2.0 2.7 0.2 1.5 4.3 3.2 5.7 1.3 1.2 2.4 242 B L 4.0 5.1 7.0 6.520 9.5 20 12.9 13.6 0 8.7 243 B F 0.7 1.8 1.2 0 0.2 4.9 1.5 4.8 1.4 0.82.3 244 B P 2.1 3.1 5.4 2.8 7.0 3.5 3.7 0.7 0 3.6 3.3 245 B P 2.1 20 2020 20 7.3 20 50 20 20 20 246 B K 1.4 2.8 1.8 2.0 3.4 5.8 3.9 2.3 1.8 1.23.2 247 B P 2.4 5.0 8.4 7.4 20 7.5 18.5 8.7 9.4 20 7.9 248 B K 2.0 4.14.6 3.7 20 6.8 9.1 3.7 0 2.2 2.9 249 B D 3.2 4.2 3.1 1.2 3.2 9.1 4.0 502.9 0 3.3 250 B T 0.1 1.4 3.1 7.4 20 6.7 20 2.9 5.9 8.9 6.1 251 B L 4.05.6 6.9 7.9 3.5 10.4 1.9 4.9 5.0 2.1 3.3 252 B M 3.0 3.6 5.6 2.9 20 9.220 6.0 1.0 4.9 0 253 B I 1.5 2.3 4.6 3.5 4.6 6.1 6.8 1.7 0 1.6 2.2 254 BS 0 2.8 6.1 3.4 8.5 4.1 9.9 4.5 4.2 1.5 3.9 255 B R 1.8 3.7 5.0 3.7 3.56.8 0.8 14.1 0.1 0 1.5 256 B T 1.0 2.3 1.6 1.4 3.1 3.7 2.3 3.5 1.2 1.92.6 257 B P 2.0 10.4 20 20 50 8.4 50 20 20 17.3 20 258 B E 0 1.6 2.7 0.80.8 3.9 2.0 12.0 2.4 4.0 1.8 259 B V 4.1 4.5 7.0 9.1 20 10.5 20 1.5 12.214.4 15.5 260 B T 2.4 2.7 3.0 0 20 7.2 7.2 1.6 3.0 5.3 3.5 261 B C 018.0 20 20 20 4.0 20 20 20 20 20 262 B V 1.6 1.5 0.8 20 20 7.0 20 8.416.8 20 20 263 B V 4.5 5.2 5.5 18.4 20 10.9 20 10.9 4.6 3.5 8.9 264 B V2.6 3.0 3.7 1.6 12.8 7.1 12.2 0 2.3 3.1 4.0 265 B D 1.4 2.7 2.4 1.3 2.02.3 2.3 50 1.0 0 2.4 266 B V 4.9 5.4 7.0 15.9 20 11.6 20 2.7 20 20 19.7267 B S 2.4 4.6 4.3 4.3 1.3 7.1 2.8 1.3 0.9 1.7 3.6 268 B H 2.1 2.9 3.82.4 6.9 5.5 2.0 1.7 0 4.9 2.7 269 B E 0.8 2.5 0.9 1.2 3.3 4.0 2.9 1.22.0 2.2 3.2 270 B N 0 1.3 0.9 3.9 2.9 3.8 4.8 5.7 3.5 1.2 3.8 271 B P0.8 2.5 6.0 7.8 3.1 5.6 4.2 16.4 5.7 16.9 6.0 272 B E 0.7 1.7 0.1 0 3.03.7 2.8 1.0 1.9 1.8 3.1 273 B V 4.8 5.5 5.1 16.6 20 10.8 20 3.1 9.6 7.29.8 274 B K 0.8 2.4 1.6 1.0 2.3 4.5 2.7 1.5 1.5 1.4 3.1 275 B F 6.9 8.59.9 8.0 0 12.1 3.7 8.2 4.8 5.5 6.8 276 B N 0.4 1.6 1.2 0 20 5.3 20 9.12.7 3.1 2.7 277 B W 8.9 10.9 11.6 10.6 4.8 14.6 7.8 10.1 6.3 7.6 7.5 278B Y 0.8 2.1 5.6 0 1.4 6.3 3.9 18.3 1.3 12.7 1.7 279 B V 3.8 4.9 5.9 3.820 8.8 16.0 1.0 0 2.8 3.6 280 B D 3.5 3.4 0 1.8 12.4 3.9 9.8 17.0 3.62.3 4.0 281 B G 50 50 50 50 50 0 50 50 50 50 50 282 B V 0.4 1.8 1.7 0.82.2 3.9 2.1 0.8 0.9 1.3 2.9 283 B E 0.9 1.2 4.9 0 7.8 4.7 4.3 1.7 1.54.7 2.0 284 B V 2.3 2.7 5.0 2.6 16.4 7.0 5.5 3.1 2.0 7.0 2.9 285 B H 0.62.0 1.8 1.0 2.4 2.5 2.3 2.0 1.4 1.9 2.9 286 B N 1.0 1.8 0.9 0.2 3.4 3.53.1 0 1.4 2.2 2.4 287 B A 2.5 4.3 5.5 8.5 0 8.4 3.2 13.3 6.7 17.7 4.6288 B K 0.6 1.7 1.6 0.8 2.5 3.7 2.5 1.0 1.4 1.7 2.0 289 B T 0.9 1.5 2.10.5 5.4 4.6 2.3 0 0.2 0.2 1.3 290 B K 0.7 2.4 2.5 1.2 3.2 5.2 5.2 2.40.2 0.1 1.8 291 B P 1.0 2.5 2.0 1.7 4.5 3.6 3.4 2.5 1.6 2.6 3.4 292 B R1.8 3.5 3.5 2.2 1.6 5.5 2.8 2.2 1.1 0 1.8 293 B E 0.7 2.2 1.6 0.6 8.64.5 4.7 2.8 1.5 0.9 2.4 294 B E 2.1 2.2 0.5 0.4 5.2 5.7 3.3 2.8 1.3 3.02.4 295 B Q 3.3 3.1 4.0 3.1 18.8 7.6 13.1 8.3 1.2 0 2.4 296 B Y 4.2 4.44.5 3.6 5.6 0 4.4 50 3.4 4.4 5.5 297 B N 1.3 2.1 3.1 1.4 4.0 4.2 2.7 0.31.3 2.1 2.3 298 B S 5.5 6.0 6.3 5.7 9.7 0 7.5 50 5.7 6.3 7.8 299 B T 1.13.1 3.5 15.2 0.9 5.9 0 6.3 1.4 10.9 2.3 300 B Y 3.1 3.7 4.0 3.8 2.9 8.82.3 10.5 2.0 1.5 2.8 301 B R 1.5 2.3 1.4 0.3 20 6.4 20 6.8 0.1 3.8 1.7302 B V 3.4 4.0 5.8 3.9 20 9.3 7.0 0.2 1.8 3.4 2.9 303 B V 0.2 0.2 1.21.2 20 5.3 20 5.8 3.9 11.4 4.9 304 B S 1.0 1.9 4.1 10.8 20 7.8 20 8.37.4 20 12.1 305 B V 1.5 1.8 1.7 3.9 20 6.2 20 0.7 3.2 4.2 2.9 306 B L5.2 7.1 6.7 7.3 1.5 11.6 4.9 3.7 5.1 0 6.0 307 B T 1.6 3.0 2.5 1.9 1.15.5 3.0 0.2 1.6 1.2 3.0 308 B V 5.1 5.8 12.7 7.4 20 11.3 20 0 19.1 6.120 309 B L 1.3 2.8 1.9 1.7 3.2 5.4 3.3 0.9 1.5 1.0 3.2 310 B H 1.7 2.42.5 3.8 13.1 6.4 5.5 3.6 3.6 7.5 3.7 311 B Q 0 1.6 1.1 0.5 0.9 3.7 1.60.4 0.6 0.6 1.8 312 B D 0 1.7 0.8 4.6 20 5.3 20 11.0 3.1 2.9 2.7 313 B W4.4 5.9 7.2 5.7 0 10.3 5.3 9.3 3.0 4.3 2.9 314 B L 2.8 4.4 5.7 3.7 207.6 8.3 4.5 1.6 0 3.0 315 B N 0 7.4 3.9 5.2 12.9 2.1 12.0 14.7 4.5 3.35.6 316 B G 8.9 7.7 9.2 6.9 13.3 0 10.8 50 6.7 6.8 7.0 317 B K 2.6 4.37.6 5.9 6.7 7.4 2.3 7.5 0 4.8 3.4 318 B E 1.7 2.6 2.9 1.6 20 6.0 9.6 1.82.2 6.1 3.7 319 B Y 6.9 7.8 9.3 10.1 0 12.7 4.7 3.1 6.2 7.1 6.9 320 B K1.7 2.9 6.7 1.9 20 7.0 20 0.6 0 8.9 2.0 321 B C 0 4.5 20 20 20 6.2 20 2020 20 20 322 B K 2.8 3.8 6.2 3.3 20 8.4 20 1.8 0.8 16.3 3.0 323 B V 3.54.4 8.8 7.6 20 9.8 20 5.4 9.2 20 6.0 324 B S 0.5 2.5 1.4 0.6 0.4 5.2 2.73.5 0 0.2 2.4 325 B N 4.1 5.5 6.9 5.7 20 9.6 20 1.0 10.5 11.2 17.0 326 BK 0.9 2.8 1.8 1.8 2.6 4.4 2.5 4.2 1.3 2.9 3.5 327 B A 3.1 4.4 6.1 3.210.3 7.4 4.0 7.0 2.8 2.5 4.5 328 B L 4.7 5.4 6.6 3.3 20 9.8 20 50 4.1 015.1 329 B P 0.6 2.1 0.9 1.0 3.8 3.2 2.8 2.2 1.3 2.4 3.2 330 B A 0.4 1.81.5 1.2 3.4 3.3 2.7 2.1 1.3 1.9 3.0 331 B P 1.6 3.6 7.8 10.6 7.3 6.5 4.68.9 7.7 13.6 6.3 332 B I 2.0 3.0 2.6 0.9 5.8 6.9 2.6 0 2.1 0.1 2.5 333 BE 2.2 2.8 2.7 0 8.1 6.4 7.8 3.4 3.6 6.1 3.3 334 B K 2.2 3.4 4.2 2.1 10.47.2 4.1 1.7 1.8 3.1 2.9 335 B T 0.5 1.1 1.9 0.8 4.8 4.6 4.4 0.5 0.3 3.72.2 336 B I 0.7 1.5 2.5 1.0 18.4 5.0 5.4 0.1 1.9 4.6 1.9 337 B S 0.4 1.14.9 10.6 20 3.4 20 50 7.9 11.0 5.1 338 B K 4.5 8.2 7.4 8.0 20 9.6 20 5.30 2.0 5.7 339 B A 1.5 2.8 3.0 2.1 2.4 5.5 3.3 0 0.4 0.2 2.8 340 B K 1.02.7 1.7 1.3 1.7 3.7 2.1 2.4 0 2.1 1.9 Pos WT N P Q R S T V W Y 237 A G2.5 0.5 1.3 1.5 0 1.2 1.9 7.5 5.6 238 A P 7.3 0 6.1 20 1.3 3.7 2.1 20 20239 A S 0.2 8.4 0.9 0.5 0.3 1.3 2.5 4.7 1.8 240 A V 2.2 0.3 7.4 20 2.0 00.4 20 20 241 A F 0.7 10 0.7 1.4 0 0.9 6.9 3.9 0.9 242 A L 5.7 3.6 9.220 4.8 4.7 3.4 20 20 243 A F 0.3 50 0 0.5 0.5 0.5 4.0 4.3 0.6 244 A P3.8 0.3 1.1 0.9 1.4 1.5 0.9 7.7 6.9 245 A P 20 0 20 20 3.4 19.2 50 20 20246 A K 0 1.2 2.7 2.3 2.2 2.5 2.9 6.8 3.7 247 A P 9.5 0 7.0 8.4 0.1 0.64.6 20 50 248 A K 4.1 2.4 3.1 3.0 2.1 0.8 2.0 15.2 20 249 A D 3.1 50 1.94.1 4.4 8.1 50 6.3 5.3 250 A T 2.2 50 7.3 10.7 1.6 0.8 0 20 20 251 A L4.7 50 5.2 4.4 4.8 2.1 2.4 50 0 252 A M 4.7 50 2.9 14.2 0.9 6.7 6.0 2020 253 A I 2.3 50 2.6 2.7 1.2 1.2 2.0 5.8 4.7 254 A S 2.1 50 4.4 4.6 0.25.1 6.0 9.0 8.9 255 A R 3.2 50 2.0 0.6 2.0 1.4 14.5 5.4 20 256 A T 0 2.30.4 0.3 0.7 1.6 2.4 6.0 3.0 257 A P 20 0 18.6 20 4.1 20 20 50 50 258 A E1.9 14.7 2.2 2.7 0.9 4.1 10.4 20 1.0 259 A V 7.7 50 12.2 20 5.1 1.8 0 2020 260 A T 2.7 4.5 1.1 3.0 2.5 2.0 2.0 14.0 20 261 A C 20 50 20 20 4.220 20 20 20 262 A V 3.6 50 20 16.5 2.1 1.9 0 20 20 263 A V 5.7 50 14.418.7 6.0 3.6 0 20 20 264 A V 2.3 10.4 1.9 2.4 2.6 1.6 0.5 15.9 18.2 265A D 1.1 50 0.5 0.8 1.6 11.8 19.0 4.9 2.1 266 A V 5.4 50 19.9 20 6.3 4.20 50 20 267 A S 3.7 0 2.2 1.5 1.9 1.3 2.6 5.6 2.3 268 A H 0.6 0.5 0.91.0 0.7 1.8 1.6 7.3 4.6 269 A E 0 6.8 0.5 1.5 0.1 0.3 1.3 5.3 2.7 270 AN 1.6 13.3 3.2 3.0 0.9 1.2 5.1 2.2 1.0 271 A P 4.2 0 8.0 4.2 1.3 2.9 7.54.3 2.6 272 A E 0.3 11.8 0.2 1.6 0 0.9 1.2 4.4 2.8 273 A V 1.2 50 20 201.4 0 0.7 20 20 274 A K 0.9 50 0 0.6 0.6 1.1 1.4 2.8 2.2 275 A F 8.3 7.07.1 11.9 8.1 7.3 7.4 10.3 1.8 276 A N 0.2 18.7 1.3 1.8 1.1 6.3 10.8 2020 277 A W 11.4 7.6 9.5 14.6 10.2 9.6 11.3 0 20 278 A Y 3.3 50 0.3 2.20.5 0 10 8.6 1.0 279 A V 3.8 20 0.5 3.6 3.8 2.3 1.6 20 20 280 A D 0.5 503.1 3.5 3.1 8.9 13.1 13.7 13.0 281 A G 50 50 50 50 50 19.2 50 50 50 282A V 0.4 50 0.4 0.5 0.1 0.5 0 5.4 2.5 283 A E 3.1 0.5 0.5 1.3 0.6 1.6 3.08.3 7.2 284 A V 2.7 15.1 2.4 1.6 2.6 1.3 0 11.1 16.1 285 A H 0 1.8 0.90.9 0.3 0.5 2.3 5.1 2.3 286 A N 0.2 6.6 0.4 0.9 0.7 0.7 0.8 5.4 3.4 287A A 1.3 12.9 8.3 8.6 4.1 4.5 9.9 1.4 2.0 288 A K 0.4 5.0 0.5 0.6 0 0.51.8 6.0 2.6 289 A T 1.2 0.8 0.6 0.7 0 0.2 0.9 6.2 7.0 290 A K 1.5 50 1.01.3 0.6 0 2.9 6.3 4.5 291 A P 0 0.9 0.9 1.3 0 1.0 2.4 7.6 4.1 292 A R1.9 0.9 1.3 1.6 1.5 1.6 2.5 4.3 1.9 293 A E 1.2 16.3 0 2.2 0.1 0.8 3.310.6 8.3 294 A E 0 1.6 0.9 1.3 1.9 1.6 2.4 9.0 5.4 295 A Q 3.3 50 1.63.6 3.8 7.9 10.1 20 20 296 A Y 1.7 50 2.1 2.5 1.8 14.0 50 7.7 4.7 297 AN 1.1 50 0.9 0 0.8 1.1 1.5 7.3 4.3 298 A S 5.2 50 6.2 6.3 4.4 12.0 5012.5 10.6 299 A T 0.1 50 20 3.4 0.7 0 0.4 50 1.7 300 A Y 4.4 50 5.4 7.95.7 7.2 12.7 5.8 0.4 301 A R 0.5 50 0.3 0.9 2.5 3.1 9.8 20 20 302 A V3.6 10.8 2.9 3.9 3.6 2.1 0 20 20 303 A V 2.7 1.6 4.8 4.9 1.3 0.7 1.0 2020 304 A S 2.8 20 11.2 9.4 0 2.5 4.1 20 20 305 A V 0.6 12.6 4.5 3.8 1.81.0 0 20 20 306 A L 6.0 15.0 7.3 15.4 5.9 4.2 5.6 20 2.5 307 A T 1.8 01.6 2.4 1.6 1.0 0.8 9.5 1.4 308 A V 10.2 50 13.4 20 4.4 4.4 1.7 20 20309 A L 0 0.3 1.3 0.4 1.6 1.3 0.8 5.8 3.4 310 A H 1.1 0 3.7 7.5 0.7 0.53.3 14.8 18.9 311 A Q 0 0.7 0.9 1.2 0.5 0 1.2 2.6 1.8 312 A D 1.1 50 3.03.0 0.6 5.5 9.6 16.0 20 313 A W 6.6 50 4.8 6.3 5.7 4.5 9.9 1.3 0.9 314 AL 4.0 50 3.2 2.5 3.9 3.6 5.7 17.5 20 315 A N 1.9 50 3.3 4.6 1.7 8.9 5013.0 12.0 316 A G 9.5 50 8.8 9.2 9.2 50 50 11.9 16.6 317 A K 5.0 50 3.61.5 3.1 5.2 6.8 20 7.4 318 A E 2.2 17.5 1.6 2.7 1.1 0 3.4 20 20 319 A Y8.2 20 8.9 12.7 7.7 5.8 3.8 8.7 0.9 320 A K 4.2 8.4 1.7 1.4 1.5 0.6 1.020 20 321 A C 20 20 20 20 2.2 8.6 18.8 20 20 322 A K 4.1 50 2.8 3.0 3.11.6 0 20 20 323 A V 8.7 50 9.3 20 4.9 2.5 0 20 20 324 A S 0.4 50 1.0 2.60.7 1.3 1.3 8.7 0.3 325 A N 0 13.3 6.3 20 5.1 3.7 3.4 20 20 326 A K 1.10 1.7 2.1 1.5 4.5 9.4 5.1 3.3 327 A A 3.8 20 3.2 2.8 1.8 0 3.3 18.3 12.6328 A L 4.3 50 1.7 6.0 2.9 8.3 5.9 50 50 329 A P 0.1 0.3 0.6 1.4 0 1.12.2 6.6 3.7 330 A A 0.3 20 0.5 1.1 0 0.1 2.2 6.0 3.4 331 A P 6.1 0 8.07.9 1.4 6.4 4.6 6.5 6.8 332 A I 2.8 50 0 3.7 2.8 1.8 1.3 12.6 9.4 333 AE 2.9 3.6 1.1 2.9 1.7 1.2 4.8 8.0 8.4 334 A K 2.5 2.8 1.5 0 2.8 1.6 1.54.1 7.9 335 A T 1.2 2.1 1.0 0.2 0.8 0 0.9 5.1 4.8 336 A I 1.3 20 0.1 2.61.1 0.4 0.3 12.3 13.9 337 A S 5.0 12.7 6.7 7.3 0 10.5 50 20 20 338 A K5.5 5.8 6.4 4.6 5.8 6.8 6.9 20 20 339 A A 1.5 1.0 0.8 0.4 1.3 1.2 0.46.6 2.4 340 A K 1.0 1.0 0 1.1 0.6 1.2 3.1 7.8 2.2 237 B G 0.5 0.3 0 0.30.4 0.3 1.0 7.3 5.4 238 B P 7.3 0 7.8 20 1.9 4.7 2.7 20 20 239 B S 0.29.3 0.8 0.3 0.4 1.7 2.7 4.8 2.1 240 B V 2.7 1.3 9.7 20 2.8 0.6 0.7 20 20241 B F 1.5 8.5 1.2 1.9 0 1.3 5.3 4.3 1.3 242 B L 5.9 3.9 10.6 19.9 5.05.6 4.2 20 20 243 B F 0.4 50 0 0.5 0.3 0.6 4.5 4.3 0.7 244 B P 3.5 0.31.0 1.2 1.2 1.4 0.5 7.3 7.2 245 B P 20 0 20 20 3.0 19.4 20 20 20 246 B K0 0.3 2.1 1.5 1.5 1.7 1.9 6.0 3.1 247 B P 12.4 0 6.3 9.1 0.9 0.8 3.0 2020 248 B K 3.8 2.1 2.7 2.5 2.3 1.2 2.1 20 20 249 B D 3.8 50 1.6 4.1 4.48.4 50 6.6 4.6 250 B T 2.2 50 6.7 6.1 1.3 0.6 0 20 20 251 B L 5.3 50 9.35.4 5.9 3.3 4.5 50 0 252 B M 4.2 50 2.4 14.0 1.5 6.9 7.7 20 20 253 B I3.0 50 3.3 2.4 1.6 1.5 2.3 6.5 5.3 254 B S 3.1 50 3.4 5.1 0.3 5.6 6.6 108.7 255 B R 2.6 50 1.6 0 1.7 1.0 13.6 4.8 20 256 B T 0 2.4 0.5 0.2 0.71.5 2.8 5.9 2.9 257 B P 20 0 16.5 20 4.2 20 20 50 50 258 B E 2.1 16.22.9 3.0 0.7 4.5 11.7 20 1.6 259 B V 7.5 50 8.8 20 5.3 2.0 0 20 20 260 BT 2.7 4.1 0.3 1.3 2.5 2.0 1.6 15.9 20 261 B C 20 50 20 20 3.5 20 20 2020 262 B V 3.5 50 14.9 19.5 1.9 1.6 0 20 20 263 B V 4.7 50 15.3 19.2 5.73.1 0 20 20 264 B V 2.5 5.0 1.5 3.1 2.4 1.6 0.4 20 20 265 B D 0.9 50 0.30.7 1.6 11.4 18.0 5.0 2.2 266 B V 6.2 50 17.2 20 5.8 4.1 0 50 50 267 B S2.7 0 1.4 1.9 2.3 1.8 3.2 5.2 2.5 268 B H 1.8 3.2 0.9 1.1 1.4 1.2 2.88.4 6.4 269 B E 0 5.3 0.8 1.6 0.8 1.0 1.8 5.6 3.1 270 B N 2.9 15.5 4.03.5 1.0 1.4 7.0 6.6 3.1 271 B P 4.0 0 7.6 5.3 1.3 2.9 6.2 5.0 3.6 272 BE 0.6 17.4 0.1 1.0 0.3 1.0 0.6 3.7 3.0 273 B V 4.2 50 20 20 1.5 0 2.5 2020 274 B K 0.6 50 0 0.5 0.9 1.3 1.1 2.8 2.4 275 B F 8.0 7.1 7.2 13.0 8.27.1 7.4 10.7 2.0 276 B N 0.1 19.6 1.1 1.7 0.5 5.9 8.8 20 20 277 B W 10.96.8 9.3 14.0 10 9.5 11.0 0 20 278 B Y 3.0 18.1 0.3 2.4 1.2 0.3 10.3 7.50.3 279 B V 4.5 20 1.0 4.1 4.5 2.9 2.0 20 20 280 B D 0.2 50 3.1 3.6 3.08.8 12.1 13.9 12.7 281 B G 50 50 50 50 50 50 50 50 50 282 B V 0.2 50 0.30.3 0 0.3 0 5.6 2.4 283 B E 3.2 0.4 0.7 0.8 0.6 1.5 2.7 8.3 7.5 284 B V3.2 50 2.6 2.2 2.7 1.4 0 13.3 20 285 B H 0 1.6 0.5 0.7 0 0.2 2.2 4.8 1.9286 B N 0.2 8.3 0.4 1.0 0.7 0.8 1.0 5.4 3.4 287 B A 1.1 14.1 8.2 9.1 3.53.2 10.7 1.1 1.4 288 B K 0.3 5.7 0.6 0.4 0 0.5 1.7 5.8 2.3 289 B T 1.10.7 0.5 0.2 0.2 0.2 0.4 6.7 5.4 290 B K 1.3 50 0.6 1.2 0.7 0 2.4 5.7 4.3291 B P 0.2 0.8 0.7 1.2 0 0.8 2.6 7.6 3.9 292 B R 2.4 1.3 1.6 1.5 1.82.0 2.6 4.5 2.0 293 B E 1.1 15.9 0 2.3 0.2 0.9 2.8 9.3 8.6 294 B E 0 1.30.9 1.3 1.5 1.3 2.2 8.4 6.2 295 B Q 3.4 50 1.8 3.8 3.6 7.2 8.7 20 18.4296 B Y 2.5 50 2.9 3.3 2.6 11.3 50 8.5 5.4 297 B N 0.9 50 1.0 0 0.8 0.81.3 7.0 4.1 298 B S 4.5 50 5.5 5.4 3.3 9.6 50 11.9 9.6 299 B T 0.9 5015.0 1.8 1.8 1.1 2.3 20 1.6 300 B Y 3.2 50 4.3 5.9 4.6 3.1 10.9 5.7 0301 B R 0.7 50 0 0.6 2.4 2.7 7.1 20 20 302 B V 4.8 20 2.8 5.0 3.7 2.1 020 20 303 B V 2.3 0.9 5.3 6.4 0.4 0 0.2 20 20 304 B S 2.6 16.4 14.1 12.00 1.7 3.6 20 20 305 B V 0.7 14.7 4.2 3.3 1.7 0.9 0 20 20 306 B L 6.012.2 6.9 14.6 6.2 4.5 5.5 20 1.9 307 B T 2.1 0 1.7 2.3 1.7 1.1 0.8 9.71.5 308 B V 11.7 50 10.8 20 5.6 4.5 2.5 20 20 309 B L 0 0.2 1.2 0.4 1.61.3 0.8 5.8 3.3 310 B H 1.2 0 4.1 10.5 1.2 0.6 4.6 11.6 13.6 311 B Q 01.7 0.8 1.0 0.4 0 1.4 2.3 1.0 312 B D 1.5 50 6.8 4.0 0.5 7.1 9.7 20 20313 B W 7.7 50 5.1 7.1 6.0 4.8 7.2 1.6 1.4 314 B L 4.0 50 3.4 4.0 3.93.6 5.9 17.5 20 315 B N 2.5 50 5.8 5.9 2.0 11.4 18.0 13.7 11.8 316 B G7.5 50 6.8 7.7 6.9 50 50 10 13.8 317 B K 4.4 50 3.2 1.3 2.2 6.6 6.4 207.6 318 B E 2.4 13.4 1.7 3.0 1.1 0 3.4 19.3 20 319 B Y 8.0 50 9.5 13.17.4 5.5 3.6 10.4 0.8 320 B K 3.9 12.6 1.8 1.2 1.6 0.7 1.1 20 20 321 B C20 20 20 20 2.3 9.1 19.2 20 20 322 B K 4.6 50 2.7 2.7 3.5 2.0 0 20 20323 B V 9.2 50 9.8 19.4 4.8 2.7 0 20 20 324 B S 1.3 11.1 1.0 2.7 0.9 1.234 2.5 1.2 325 B N 0.4 11.5 5.5 16.5 3.1 0 2.1 20 50 326 B K 0.1 0 1.21.3 0.7 2.4 3.4 6.4 2.4 327 B A 3.9 20 3.5 3.7 1.4 0 2.8 10.3 12.2 328 BL 6.0 50 3.7 6.2 5.5 18.4 50 20 50 329 B P 0 0.4 0.3 0.9 0 0.6 1.6 6.43.7 330 B A 0 20 0.6 1.3 0.1 0.3 1.8 6.0 3.7 331 B P 5.7 0 8.3 7.7 1.75.3 5.2 6.5 8.0 332 B I 2.6 50 0 2.4 2.3 1.3 0.9 15.3 6.6 333 B E 2.63.6 1.2 3.0 2.4 1.4 4.9 8.4 8.8 334 B K 2.6 2.8 1.5 0 2.7 1.6 1.4 5.510.8 335 B T 0.8 1.7 1.7 0.6 0.6 0 0.8 5.2 5.1 336 B I 1.5 20 0 3.0 1.50.5 0.4 14.2 19.6 337 B S 3.6 12.8 6.2 7.0 0 4.6 50 20 20 338 B K 6.05.8 6.8 4.9 5.4 6.4 6.7 20 20 339 B A 1.4 1.5 0.9 0.7 1.5 1.1 0.5 6.62.6 340 B K 0.6 1.0 0 1.0 0.2 1.0 2.3 7.0 1.8SPA™ technology; 1DN2 template structure; +carbohydrate

The results of the design calculations presented above in Tables 1-60were used to construct a series of Fc variant libraries for experimentalproduction and screening. Experimental libraries were designed insuccessive rounds of computational and experimental screening. Design ofsubsequent Fc libraries benefitted from feedback from prior libraries,and thus typically comprised combinations of Fc variants that showedfavorable properties in the previous screen. The entire set of Fcvariants that were constructed and experimentally tested is shown inTable 61. In this table, row 1 lists the variable positions, and therows that follow indicate the amino acids at those variable positionsfor WT and the Fc variants. For example, variant 18 has the followingfour mutations: F241E, F243Y, V262T, and V264R. The variable positionresidues that compose this set of Fc variants are illustratedstructurally in FIG. 4, and are presented in the context of the humanIgG1 Fc sequence in FIG. 5. TABLE 61 Variant Substitution(s) 1 V264A 2V264L 3 V264I 4 F241W 5 F241L 6 F243W 7 F243L 8 F241L/F243L/V262I/V264I9 F241W/F243W 10 F241W/F243W/V262A/V264A 11 F241L/V262I 12 F243L/V264I13 F243L/V262I/V264W 14 F241Y/F243Y/V262T/V264T 15F241E/F243R/V262E/V264R 16 F241E/F243Q/V262T/V264E 17F241R/F243Q/V262T/V264R 18 F241E/F243Y/V262T/V264R 19 L328M 20 L328E 21L328F 22 I332E 23 L328M/I332E 24 P244H 25 P245A 26 P247V 27 W313F 28P244H/P245A/P247V 29 P247G 30 V264I/I332E 31F241E/F243R/V262E/V264R/I332E 32 F241E/F243Q/V262T/V264E/I332E 33F241R/F243Q/V262T/V264R/I332E 34 F241E/F243Y/V262T/V264R/I332E 35 S298A36 S298A/I332E 37 S298A/E333A/K334A 41 S239E/I332E 42 S239Q/I332E 43S239E 44 D265G 45 D265N 46 S239E/D265G 47 S239E/D265N 48 S239E/D265Q 49Y296E 50 Y296Q 51 S298T 52 S298N 53 T299I 54 A327S 55 A327N 56S267Q/A327S 57 S267L/A327S 58 A327L 59 P329F 60 A330L 61 A330Y 62 I332D63 N297S 64 N297D 65 N297S/I332E 66 N297D/I332E 67 N297E/I332E 68D265Y/N297D/I332E 69 D265Y/N297D/T299L/I332E 70 D265F/N297E/I332E 71L328I/I332E 72 L328Q/I332E 73 I332N 74 I332Q 75 V264T 76 V264F 77 V240I78 V263I 79 V266I 80 T299A 81 T299S 82 T299V 83 N325Q 84 N325L 85 N325I86 S239D 87 S239N 88 S239F 89 S239D/I332D 90 S239D/I332E 91 S239D/I332N92 S239D/I332Q 93 S239E/I332D 94 S239E/I332N 95 S239E/I332Q 96S239N/I332D 97 S239N/I332E 98 S239N/I332N 99 S239N/I332Q 100 S239Q/I332D101 S239Q/I332N 102 S239Q/I332Q 103 K326E 104 Y296D 105 Y296N 106N297D/I332E/F241Y/F243Y/V262T/V264T 107 I332E/A330Y 108I332E/V264I/A330Y 109 I332E/A330L 110 I332E/V264I/A330L 111 L234D 112L234E 113 L234N 114 L234Q 115 L234T 116 L234H 117 L234Y 118 L234I 119L234V 120 L234F 121 L235D 122 L235S 123 L235N 124 L235Q 125 L235T 126L235H 127 L235Y 128 L235I 129 L235V 130 L235F 131 S239T 132 S239H 133S239Y 134 V240A 135 V240T 136 V240M 137 V263A 138 V263T 139 V263M 140V264M 141 V264Y 142 V266A 143 V266T 144 V266M 145 E269H 146 E269Y 147E269F 148 E269R 149 Y296S 150 Y296T 151 Y296L 152 Y296I 153 S298H 154T299H 155 A330V 156 A330I 157 A330F 158 A330R 159 A330H 160 N325D 161N325E 162 N325A 163 N325T 164 N325V 165 N325H 166 L328D/I332E 167L328E/I332E 168 L328N/I332E 169 L328Q/I332E 170 L328V/I332E 171L328T/I332E 172 L328H/I332E 173 L328I/I332E 174 L328A 175 I332T 176I332H 177 I332Y 178 I332A 179 V264I/I332E/S239E 180 V264I/I332E/S239Q181 V264I/I332E/S239E/A330Y 182 V264I/I332E/S239E/A330Y/S298A 183N297D/I332E/S239D 184 N297D/I332E/S239E 185 N297D/I332E/S239D/D265V 186N297D/I332E/S239D/D265I 187 N297D/I332E/S239D/D265L 188N297D/I332E/S239D/D265F 189 N297D/I332E/S239D/D265Y 190N297D/I332E/S239D/D265H 191 N297D/I332E/S239D/D265T 192N297D/I332E/V264E 193 N297D/I332E/Y296D 194 N297D/I332E/Y296E 195N297D/I332E/Y296N 196 N297D/I332E/Y296Q 197 N297D/I332E/Y296H 198N297D/I332E/Y296T 199 N297D/I332E/T299V 200 N297D/I332E/T299I 201N297D/I332E/T299L 202 N297D/I332E/T299F 203 N297D/I332E/T299H 204N297D/I332E/T299E 205 N297D/I332E/A330Y 206 N297D/I332E/S298A/A330Y 207S239D/I332E/A330Y 208 S239N/I332E/A330Y 209 S239D/I332E/A330L 210S239N/I332E/A330L 211 I332E/V264I/S298A 212 I332E/S239D/S298A 213I332E/S239N/S298A 214 S239D/I332E/V264I 215 S239D/I332E/V264I/S298A 216S239D/I332E/V264I/A330L 217 L328N 218 L328H 219 S239D/I332E/A330I 220N297D/I332E/S239D/A330L 221 P230A 222 E233D 223 P230A/E233D 224P230A/E233D/I332E 225 S267T 226 S267H 227 S267D 228 S267N 229 E269T 230E269L 231 E269N 232 D270Q 233 D270T 234 D270H 235 E272S 236 E272K 237E272I 238 E272Y 239 V273I 240 K274T 241 K274E 242 K274R 243 K274L 244K274Y 245 F275W 246 N276S 247 N276E 248 N276R 249 N276L 250 N276Y 251Y278T 252 Y278E 253 Y278K 254 Y278W 255 E283R 256 V302I 257 E318R 258K320T 259 K320D 260 K320I 261 K322T 262 K322H 263 V323I 264 S324T 265S324D 266 S324R 267 S324I 268 S324V 269 S324L 270 S324Y 271 K326L 272K326I 273 K326T 274 A327D 275 A327T 276 A330S 277 A330W 278 A330M 279P331V 280 P331H 281 E333T 282 E333H 283 E333I 284 E333Y 285 K334I 286K334T 287 K334F 288 T335D 289 T335R 290 T335Y 291 L234I/L235D 292V240I/V266I 293 S239D/A330Y/I332E/L234I 294 S239D/A330Y/I332E/L235D 295S239D/A330Y/I332E/V240I 296 S239D/A330Y/I332EA/264T 297S239D/A330Y/I332E/V266I 298 S239D/A330Y/I332E/K326E 299S239D/A330Y/I332E/K326T 300 S239D/N297D/I332E/A330Y 301S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T 302S239D/N297D/I332E/L235D 303 S239D/N297D/I332E/K326E

Example 2 Experimental Production and Screening of Fc Libraries

The majority of experimentation on the Fc variants was carried out inthe context of the anti-cancer antibody alemtuzumab (Campath®, aregistered trademark of Ilex Pharmaceuticals LP). Alemtuzumab binds ashort linear epitope within its target antigen CD52 (Hale et al., 1990,Tissue Antigens 35:118-127; Hale, 1995, Immunotechnology 1:175-187).Alemtuzumab has been chosen as the primary engineering template becauseits efficacy is due in part to its ability to recruit effector cells(Dyer et al., 1989, Blood 73:1431-1439; Friend et al., 1991, TransplantProc 23:2253-2254; Hale et al., 1998, Blood 92:4581-4590; Glennie etal., 2000, Immunol Today 21:403-410), and because production and use ofits antigen in binding assays are relatively straightforward. In orderto evaluate the optimized Fc variants of the present invention in thecontext of other antibodies, select Fc variants were evaluated in theanti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDECPharmaceuticals Corporation), the anti-Her2 antibody trastuzumab(Herceptin®, a registered trademark of Genentech), and the anti-EGFRantibody cetuximab (Erbitux®, a registered trademark of Imclone). Theuse of alemtuzumab, rituximab, and trastuzumab for screening purposes isnot meant to constrain the present invention to any particular antibody.

The IgG1 full length light (V_(L)-C_(L)) and heavy (V_(H)-Cγ1-Cγ2-Cγ3)chain antibody genes for alemtuzumab, rituximab, and trastuzumab wereconstructed with convenient end restriction sites to facilitatesubcloning. The genes were ligated into the mammalian expression vectorpcDNA3.1Zeo (Invitrogen). The V_(H)-Cγ1-Cγ2-Cγ3 clone in pcDNA3.1zeo wasused as a template for mutagenesis of the Fc region. Mutations wereintroduced into this clone using PCR-based mutagenesis techniques. Fcvariants were sequenced to confirm the fidelity of the sequence.Plasmids containing heavy chain gene (V_(H)-Cγ1-Cγ2-Cγ3) (wild-type orvariants) were co-transfected with plasmid containing light chain gene(V_(L)-C_(L)) into 293T cells. Media were harvested 5 days aftertransfection. Expression of immunoglobulin was monitored by screeningthe culture supernatant of transfectomas by western usingperoxidase-conjugated goat-anti human IgG (Jackson ImmunoResearch,catalog # 109-035-088). FIG. 6 shows expression of wild-type alemtuzumaband variants 1 through 10 in 293T cells. Antibodies were purified fromthe supernatant using protein A affinity chromatography (Pierce, Catalog# 20334. FIG. 7 shows results of the protein purification for WTalemtuzumab. Antibody Fc variants showed similar expression andpurification results to WT. Some Fc variants were deglycosylated inorder to determine their solution and functional properties in theabsence of carbohydrate. To obtain deglycosylated antibodies, purifiedalemtuzumab antibodies were incubated with peptide-N-glycosidase (PNGaseF) at 37° C. for 24 h. FIG. 8 presents an SDS PAGE gel confirmingdeglycosylation for several Fc variants and WT alemtuzumab.

In order to confirm the functional fidelity of alemtuzumab producedunder these conditions, the antigenic CD52 peptide, fused to GST, wasexpressed in E.coli BL21 (DE3) under IPTG induction. Both un-induced andinduced samples were run on a SDS PAGE gel, and transferred to PVDFmembrane. For western analysis, either alemtuzumab from Sotec (finalconcentration 2.5 ng/ul) or media of transfected 293T cells (finalalemtuzumab concentration about 0.1-0.2 ng/ul) were used as primaryantibody, and peroxidase-conjugated goat-anti human IgG was used assecondary antibody. FIG. 9 presents these results. The ability to bindtarget antigen confirms the structural and functional fidelity of theexpressed alemtuzumab. Fc variants that have the same variable region asWT alemtuzumab are anticipated to maintain a comparable binding affinityfor antigen.

In order to screen for Fc/FcγR binding, the extracellular regions ofhuman V158 FcγRIIIa, human F158 FcγRIIIa, human FcγRIIb, human FcγRIIa,and mouse FcγRIII, were expressed and purified. FIG. 10 presents an SDSPAGE gel that shows the results of expression and purification of humanV158 FcγRIIIa. The extracellular region of this receptor was obtained byPCR from a clone obtained from the Mammalian Gene Collection(MGC:22630). The receptor was fused with glutathione S-Transferase (GST)to enable screening. Tagged FcγRIIIa was transfected in 293T cells, andmedia containing secreted FcγRIIIa were harvested 3 days later andpurified. For western analysis, membrane was probed with anti-GSTantibody.

Binding affinity to FcγRIIIa and FcγRIIb was measured for all designedFc variants using an AlphaScreen™ assay (Amplified Luminescent ProximityHomogeneous Assay (ALPHA), PerkinElmer, Wellesley, Mass.), a bead-basednon-radioactive luminescent proximity assay. Laser excitation of a donorbead excites oxygen, which if sufficiently close to the acceptor beadgenerates a cascade of chemiluminescent events, ultimately leading tofluorescence emission at 520-620 nm. The AlphaScreen™ assay was appliedas a competition assay for screening Fc variants. WT alemtuzumabantibody was biotinylated by standard methods for attachment tostreptavidin donor beads, and GST-tagged FcγR was bound to glutathionechelate acceptor beads. In the absence of competing Fc variants, WTantibody and FcγR interact and produce a signal at 520-620 nm. Additionof untagged Fc variant competes with the WT Fc/FcγR interaction,reducing fluorescence quantitatively to enable determination of relativebinding affinities. All Fc variants were screened for V158 FcγRIIIabinding using the AlphaScreen™ assay. Fc variants were screened in thecontext of either alemtuzumab or trastuzumab, and select Fc variantswere also screened in the context of rituximab and cetuximab. Select Fcvariants were subsequently screened for binding to FcγRIIb, as well asother FcγRs and Fc ligands.

FIG. 11 shows AlphaScreen™ data for binding to human V158 FcγRIIIa byselect Fc variants. The binding data were normalized to the maximum andminimum luminescence signal for each particular curve, provided by thebaselines at low and high antibody concentrations respectively. The datawere fit to a one site competition model using nonlinear regression, andthese fits are represented by the curves in the figure. These fitsprovide the inhibitory concentration 50% (IC50) (i.e. the concentrationrequired for 50% inhibition) for each antibody, illustrated by thedotted lines in FIG. 11, thus enabling the relative binding affinitiesof Fc variants to be quantitatively determined. Here, WT alemtuzumab hasan IC50 of (4.63×10⁻⁹)×(2)=9.2 nM, whereas S239D has an IC50 of(3.98×10⁻¹⁰)×(2)=0.8 nM. Thus S239D alemtuzumab binds 9.2 nM/0.8nM=11.64-fold more than WT alemtuzumab to human V158 FcγRIIIa. Similarcalculations were performed for the binding of all Fc variants to humanV158 FcγRIIIa. Select Fc variants were also screened for binding tohuman FcγRIIb, and examples of these AlphaScreen™ binding data are shownin FIG. 12. Table 62 presents the fold-enhancement or fold-reductionrelative to the parent antibody for binding of Fc variants to human V158FcγRIIIa (column 3) and human FcγRIIb (column 4), as determined by theAlphaScreen™ assay. For these data, a fold above 1 indicates anenhancement in binding affinity, and a fold below 1 indicates areduction in binding affinity relative to WT Fc. Data for 1-206 and217-218 were obtained in the context of alemtuzumab, except for thoseindicated with an asterix (*), which were tested in the context oftrastuzumab. All data for 207-216 and 219-303 were obtained in thecontext of trastuzumab. TABLE 62 FcγIIIa- fold: FcγRIIIa FcγRIIb FcγIIb-Variant Substitution(s) Fold Fold fold 1 V264A 0.53 2 V264L 0.56 3 V264I1.43 4 F241W 0.29 5 F241L 0.26 6 F243W 0.51 7 F243L 0.51 8F241L/F243L/V262I/V264I 0.09 9 F241W/F243W 0.07 10F241W/F243W/V262A/V264A 0.04 11 F241L/V262I 0.06 12 F243L/V264I 1.23 13F243L/V262I/V264W 0.02 14 F241Y/F243Y/V262T/V264T 0.05 15F241E/F243R/V262E/V264R 0.05 16 F241E/F243Q/V262T/V264E 0.07 17F241R/F243Q/V262T/V264R 0.02 18 F241E/F243Y/V262T/V264R 0.05 19 L328M0.21 20 L328E 0.12 21 L328F 0.24 22 I332E 6.72 3.93 1.71 23 L328M/I332E2.60 24 P244H 0.83 25 P245A 0.25 26 P247V 0.53 27 W313F 0.88 28P244H/P245A/P247V 0.93 29 P247G 0.54 30 V264I/I332E 12.49  1.57* 7.96 31F241E/F243R/V262E/ 0.19 V264R/I332E 32 F241E/F243Q/V262T/ V264E/I332E 33F241R/F243Q/V262T/ V264R/I332E 34 F241E/F243Y/V262T/ 0.10 V264R/I332E 35S298A 2.21 36 S298A/I332E 21.73 37 S298A/E333A/K334A 2.56 41 S239E/I332E5.80 3.49 1.66 42 S239Q/I332E 6.60 4.68 1.41 43 S239E 10.16 44 D265G<0.02 45 D265N <0.02 46 S239E/D265G <0.02 47 S239E/D265N 0.02 48S239E/D265Q 0.05 49 Y296E 0.73 1.11 0.66 50 Y296Q 0.52 0.43 1.21 51S298T 0.94 <0.02  52 S298N 0.41 <0.02  53 T299I <0.02 54 A327S 0.23 0.390.59 55 A327N 0.19 1.15 0.17 56 S267Q/A327S 0.03 57 S267L/A327S <0.02 58A327L 0.05 59 P329F <0.02 60 A330L 0.73 0.38 1.92 61 A330Y 1.64 0.752.19 62 I332D 17.80 3.34 5.33 63 N297S <0.02 64 N297D <0.02 65N297S/I332E <0.02 66 N297D/I332E 0.08 <0.02  67 N297E/I332E <0.02 68D265Y/N297D/I332E <0.02 69 D265Y/N297D/T299L/I332E <0.02 70D265F/N297E/I332E <0.02 71 L328I/I332E 7.03 72 L328Q/I332E 1.54 73 I332N0.39 74 I332Q 0.37 75 V264T 2.73 76 V264F 0.16 77 V240I 3.25 78 V263I0.10 79 V266I 1.86 80 T299A 0.03 81 T299S 0.15 82 T299V <0.02 83 N325Q<0.02 84 N325L <0.02 85 N325I <0.02 86 S239D 11.64  4.47* 2.60 87 S239N<0.02 88 S239F 0.22 <0.02  89 S239D/I332D 14.10 90 S239D/I332E 56.1019.71* 2.85 91 S239D/I332N 7.19 92 S239D/I332Q 9.28 93 S239E/I332D 9.3394 S239E/I332N 11.93 95 S239E/I332Q 3.80 96 S239N/I332D 3.08 97S239N/I332E 14.21 98 S239N/I332N 0.43 99 S239N/I332Q 0.56 100S239Q/I332D 5.05 101 S239Q/I332N 0.39 102 S239Q/I332Q 0.59 103 K326E3.85 104 Y296D 0.62 105 Y296N 0.29 106 F241Y/F243Y/V262T/V264T/ 0.15N297D/I332E 107 A330Y/I332E 12.02 4.40 2.73 108 V264I/A330Y/I332E 12.003.54 3.39 109 A330L/I332E 10.34 2.03 5.09 110 V264I/A330L/I332E 11.151.79 6.23 111 L234D 0.21 112 L234E 1.34 2.21 0.61 113 L234N 0.56 1.390.40 114 L234Q 0.37 115 L234T 0.35 116 L234H 0.33 117 L234Y 1.42 1.081.31 118 L234I 1.55 1.14 1.36 119 L234V 0.38 120 L234F 0.30 121 L235D1.66 3.63 0.46 122 L235S 1.25 123 L235N 0.40 124 L235Q 0.51 125 L235T0.52 126 L235H 0.41 127 L235Y 1.19 10.15  0.12 128 L235I 1.10 0.94 1.17129 L235V 0.48 130 L235F 0.73 3.53 0.21 131 S239T 1.34 132 S239H 0.20133 S239Y 0.21 134 V240A 0.70 0.14 5.00 135 V240T 136 V240M 2.06 1.381.49 137 V263A 138 V263T 0.43 139 V263M 0.05 140 V264M 0.26 141 V264Y1.02 0.27 3.78 142 V266A <0.02 143 V266T 0.45 144 V266M 0.62 145 E269H<0.02 146 E269Y 0.12 147 E269F 0.16 148 E269R 0.05 149 Y296S 0.12 150Y296T <0.02 151 Y296L 0.22 152 Y296I 0.09 153 A298H 0.27 154 T299H <0.02155 A330V 0.43 156 A330I 1.71 0.02 85.5 157 A330F 0.60 158 A330R <0.02159 A330H 0.52 160 N325D 0.41 161 N325E <0.02 162 N325A 0.11 163 N325T1.10 164 N325V 0.48 165 N325H 0.73 166 L328D/I332E 1.34 167 L328E/I332E0.20 168 L328N/I332E <0.02 169 L328Q/I332E 0.70 170 L328V/I332E 2.06 171L328T/I332E 1.10 172 L328H/I332E <0.02 173 L328I/I332E 3.49 174 L328A0.20 175 I332T 0.72 176 I332H 0.46 177 I332Y 0.76 178 I332A 0.89 179S239E/V264I/I332E 15.46 180 S239Q/V264I/I332E 2.14 181S239E/V264I/A330Y/I332E 8.53 182 S239E/V264I/S298A/ A330Y/I332E 183S239D/N297D/I332E 0.28 184 S239E/N297D/I332E 0.06 185S239D/D265V/N297D/I332E 0.03 186 S239D/D265I/N297D/I332E 0.01 187S239D/D265L/N297D/I332E <0.02 188 S239D/D265F/N297D/I332E <0.02 189S239D/D265Y/N297D/I332E 0.02 190 S239D/D265H/N297D/I332E 0.04 191S239D/D265T/N297D/I332E <0.02 192 V264E/N297D/I332E 0.05 193Y296D/N297D/I332E 194 Y296E/N297D/I332E <0.02 195 Y296N/N297D/I332E 0.04196 Y296Q/N297D/I332E <0.02 197 Y296H/N297D/I332E <0.02 198Y296T/N297D/I332E <0.02 199 N297D/T299V/I332E <0.02 200N297D/T299I/I332E <0.02 201 N297D/T299L/I332E <0.02 202N297D/T299F/I332E <0.02 203 N297D/T299H/I332E <0.02 204N297D/T299E/I332E <0.02 205 N297D/A330Y/I332E 0.43 206N297D/S298A/A330Y/I332E 0.16 207 S239D/A330Y/I332E 129.58 208S239N/A330Y/I332E 14.22 209 S239D/A330L/I332E 138.63 7.50 18.48 210S239N/A330L/I332E 12.95 211 V264I/S298A/I332E 16.50 212S239D/S298A/I332E 295.16 6.16 47.92 213 S239N/S298A/I332E 32.14 5.156.24 214 S239D/V264I/I332E 36.58 14.39  2.54 215 S239D/V264I/S298A/I332E216 S239D/V264I/A330L/I332E 217 L328N 0.59 218 L328H <0.02 219S239D/I332E/A330I 59.1 220 N297D/I332E/S239D/A330L 221 P230A 1.09 222E233D 0.85 223 P230A/E233D 0.92 224 P230A/E233D/I332E 1.87 225 S267T 226S267H 227 S267D 228 S267N 229 E269T <0.02 230 E269L <0.02 231 E269N<0.02 232 D270Q <0.02 233 D270T <0.02 234 D270H <0.02 235 E272S 236E272K 237 E272I 238 E272Y 8.70 239 V273I 0.79 240 K274T 1.41 241 K274E6.11 242 K274R 1.41 243 K274L 1.09 244 K274Y 1.06 245 F275W 1.11 246N276S 0.41 247 N276E 0.87 248 N276R 0.66 249 N276L 1.07 250 N276Y 0.56251 Y278T 1.87 252 Y278E 0.90 253 Y278K 254 Y278W 0.41 255 E283R 0.67256 V302I 1.01 257 E318R 1.06 258 K320T 259 K320D 260 K320I 261 K322T262 K322H 263 V323I 0.83 264 S324T 265 S324D 1.07 266 S324R 0.71 267S324I 1.15 268 S324V 1.17 269 S324L <0.02 270 S324Y 0.98 271 K326L 272K326I 1.43 273 K326T 1.88 274 A327D <0.02 275 A327T 0.02 276 A330S 277A330W 278 A330M 279 P331V 280 P331H 281 E333T 0.78 282 E333H 0.75 283E333I 284 E333Y 285 K334I 286 K334T 287 K334F 288 T335D 2.79 289 T335R2.58 290 T335Y 1.56 291 L234I/L235D 0.07 292 V240I/V266I 1.72 293S239D/A330Y/I332E/L234I 22.39 294 S239D/A330Y/I332E/L235D 7.04 295S239D/A330Y/I332E/V240I 27.97 296 S239D/A330Y/I332E/V264T 17.72 297S239D/A330Y/I332E/V266I 298 S239D/A330Y/I332E/K326E 64.14 299S239D/A330Y/I332E/K326T 59.03 300 S239D/N297D/I332E/A330Y <0.02 301S239D/N297D/I332E/A330Y/ <0.02 F241S/F243H/V262T/V264T 302S239D/N297D/I332E/L235D 303 S239D/N297D/I332E/K326E

Example 3 Selectively Enhanced Binding to FcγRs

A number of promising Fc variants with optimized properties wereobtained from the FcγRIIIa and FcγRIIb screen. Table 62 provides Fcvariants that bind more tightly to FcγRIIIa, and thus are candidates forimproving the effector function of antibodies and Fc fusions. Theseinclude a number of variants that comprise substitutions at 239, 264,272, 274, 330, and 332. FIGS. 13 a and 13b show AlphaScreen™ bindingdata for some of these Fc variants. The majority of these Fc variantsprovide substantially greater FcγRIIIa binding enhancements overS298A/E333A/K334A.

Select Fc variants were screened in the context of multiple antibodiesin order to investigate the breadth of their applicability. AlphaScreen™data for binding of select Fc variants to human V158 FcγRIIIa in thecontext of trastuzumab, rituximab, and cetuximab are shown in FIGS. 14a, 14 b, 15 a, and 15 b. Together with the data for alemtuzumab in FIG.13, the results indicate consistent binding enhancements regardless ofthe antibody context, and thus that the Fc variants of the presentinvention are broadly applicable to antibodies and Fc fusions.

Fc variants have been obtained that show differentially enhanced bindingto FcγRIIIa over FcγRIIb. As discussed, optimal effector function mayresult from Fc variants wherein affinity for activating FcγRs is greaterthan affinity for the inhibitory FcγRIIb. AlphaScreen™ data directlycomparing binding to FcγRIIIa and FcγRIIb for two Fc variants with thisspecificity profile are shown in FIGS 16 a and 16 b. This concept can bedefined quantitatively as the fold-enhancement or -reduction of theactivating FγR (Table 62, column 3) divided by the fold-enhancement or-reduction of the inhibitory FcγR (Table 62, column 4), herein referredto as the FcγRIIIa-fold:FcγRIIb-fold ratio. This value is provided inColumn 5 in Table 62. Table 62 shows that Fc variants provide thisspecificity profile, with a FcγRIIIa-fold:FcγRIIb-fold ratio as high as86:1.

Some of the most promising Fc variants of the present invention forenhancing effector function have both substantial increases in affinityfor FcγRIIIa and favorable FcγRIIIa-fold:FcγRIIb-fold ratios. Theseinclude, for example, S239D/I332E (FcγRIIIa-fold=56,FcγRIIIa-fold:FcγRIIb-fold=3), S239D/A330Y/I332E (FcγRIIIa-fold=130),S239D/A330L/I332E (FcγRIIIa-fold=139, FcγRIIIa-fold:FcγRIIb-fold=18),and S239D/S298A/I332E (FcγRIIIa-fold=295,FcγRIIIa-fold:FcγRIIb-fold=48). FIG. 17 shows AlphaScreen™ binding datafor these and other Fc variants to human V158 FcγRIIIa.

Because there are a number of FcγRs that contribute to effectorfunction, it may be worthwhile to additionally screen Fc variantsagainst other receptors. FIG. 18 shows AlphaScreen™ data for binding ofselect Fc variants to human R131 FcγRIIa. As can be seen, thoseaforementioned variants with favorable binding enhancements andspecificity profiles also show enhanced binding to this activatingreceptor. The use of FcγRIIIa, FcγRIIb, and FcγRIIc for screening is notmeant to constrain experimental testing to these particular FcγRs; otherFcγRs are contemplated for screening, including but not limited to themyriad isoforms and allotypes of FcγRI, FcγRII, and FcγRIII from humans,mice, rats, monkeys, and the like, as previously described.

Taken together, the FcγR binding data provided in FIGS. 11-18 and Table62 indicate that a number of substitions at positions 234, 235, 239,240, 243, 264, 266, 272, 274, 278, 325, 328, 330, and 332 are promisingcandidates for improving the effector function of antibodies and Fcfusions. Because combinations of some of these substitutions havetypically resulted in additive or synergistic binding improvements, itis anticipated that as yet unexplored combinations of the Fc variantsprovided in Table 62 will also provide favorable results. Thus allcombinations of the Fc variants in Table 62 are contemplated. Likewise,combinations of any of the Fc variants in Table 62 with other discoveredor undiscovered Fc variants may also provide favorable properties, andthese combinations are also contemplated. Furthermore, it is anticipatedfrom these results that other substitutions at positions 234, 235, 239,240, 243, 264, 266, 325, 328, 330, and 332 may also provide favorablebinding enhancements and specificities, and thus substitutions at thesepositions other than those presented in Table 62 are contemplated.

Example 4 Reduced Binding to FcγRs

As discussed, although there is a need for greater effector function,for some antibody therapeutics, reduced or eliminated effector functionmay be desired. Several Fc variants in Table 62 substantially reduce orablate FcγR binding, and thus may find use in antibodies and Fc fusionswherein effector function is undesirable. AlphaScreen™ binding data forsome examples of such variants are shown in FIGS. 19 a and 19 b. TheseFc variants, as well as their use in combination, may find use foreliminating effector function when desired, for example in antibodiesand Fc fusions whose mechanism of action involves blocking or antagonismbut not killing of the cells bearing target antigen.

Example 5 Aglycosylated Fc Variants

As discussed, one goal of the current experiments was to obtainoptimized aglycosylated Fc variants. Several Fc variants providesignificant progress towards this goal. Because it is the site ofglycosylation, substitution at N297 results in an aglycosylated Fc.Whereas all other Fc variants that comprise a substitution at N297completely ablate FcγR binding, N297D/I332E has significant bindingaffinity for FcγRIIIa, shown in Table 62 and illustrated in FIG. 20. Theexact reason for this result is uncertain in the absence of ahigh-resolution structure for this variant, although the computationalscreening predictions suggest that it is potentially due to acombination of new favorable Fc/FcγR interactions and favorableelectrostatic properties. Indeed other electrostatic substitutions areenvisioned for further optimization of aglycosylated Fc. Table 62 showsthat other aglycosylated Fc variants such as S239D/N297D/I332E andN297D/A330Y/I332E provide binding enhancements that bring affinity forFcγRIIIa within 0.28- and 0.43-fold respectively of glycosylated WTalemtuzumab. Combinations of these variants with other Fc variants thatenhance FcγR binding are contemplated, with the goal of obtainingaglycosylated Fc variants that bind one or more FcγRs with affinity thatis approximately the same as or even better than glycosylated parent Fc.An additional set of promising Fc variants provide stability andsolubility enhancements in the absence of carbohydrate. Fc variants thatcomprise substitutions at positions 241, 243, 262, and 264, positionsthat do not mediate FγR binding but do determine the interface betweenthe carbohydrate and Fc, ablate FγR binding, presumably because theyperturb the conformation of the carbohydrate. In deglycosylated form,however, Fc variants F241 E/F243R/V262EN264R, F241 E/F243Q/V262TN264E,F241R/F243Q/V262T/V264R, and F241E/F243Y/V262T/V264R show strongerbinding to FcγRIIIa than in glycosylated form, as shown by theAlphaScreen™ data in FIG. 21. This result indicates that these are keypositions for optimization of the structure, stability, solubility, andfunction of aglycosylated Fc. Together these results suggests thatprotein engineering can be used to restore the favorable functional andsolution properties of antibodies and Fc fusions in the absence ofcarbohydrate, and pave the way for aglycosylated antibodies and Fcfusions with favorable solution properties and full functionality thatcomprise substitutions at these and other Fc positions.

Example 6 Affinity of Fc Variants for Polymorphic Forms of FcγRIIIa

As discussed above, an important parameter of Fc-mediated effectorfunction is the affinity of Fc for both V158 and F158 polymorphic formsof FcγRIIIa. AlphaScreen™ data comparing binding of select variants tothe two receptor allotypes are shown in FIG. 22 a (V158 FcγRIIIa) andFIG. 22 b (F158 FcγRIIIa). As can be seen, all variants improve bindingto both FcγRIIIa allotypes. These data indicate that those Fc variantsof the present invention with enhanced effector function will be broadlyapplicable to the entire patient population, and that enhancement toclinical efficacy will potentially be greatest for the low responsivepatient population who need it most.

The FcγR binding affinities of these Fc variants were furtherinvestigated using Surface Plasmon Resonance (SPR) (Biacore, Uppsala,Sweden). SPR is a sensitive and extremely quantitative method thatallows for the measurement of binding affinities of protein-proteininteractions, and has been used to effectively measure Fc/FcγR binding(Radaev et al., 2001, J Biol Chem 276:16478-16483). SPR thus provides anexcellent complementary binding assay to the AlphaScreen™ assay.His-tagged V158 FcγRIIIa was immobilized to an SPR chip, and WT and Fcvariant alemtuzumab antibodies were flowed over the chip at a range ofconcentrations. Binding constants were obtained from fitting the datausing standard curve-fitting methods. Table 63 presents dissociationconstants (Kd) for binding of select Fc variants to V158 FcγRIIIa andF158 FcγRIIIa obtained using SPR, and compares these with IC50s obtainedfrom the AlphaScreen™ assay. By dividing the Kd and IC50 for eachvariant by that of WT alemtuzumab, the fold-improvements over WT (Fold)are obtained. TABLE 63 SPR V158 SPR F158 AlphaScreen ™ AlphaScreen ™FcγRIIIa FcγRIIIa V158 FcγRIIIa F158 FcγRIIIa Kd Kd IC50 IC50 (nM) Fold(nM) Fold (nM) Fold (nM) Fold WT 68 730 6.4 17.2 V264I 64 1.1 550 1.34.5 1.4 11.5 1.5 I332E 31 2.2 72 10.1 1.0 6.4 2.5 6.9 V264I/I332E 17 4.052 14.0 0.5 12.8 1.1 15.6 S298A 52 1.3 285 2.6 2.9 2.2 12.0 1.4S298A/E333A/ 39 1.7 156 4.7 2.5 2.6 7.5 2.3 K334A

The SPR data corroborate the improvements to FcγRIIIa affinity observedby AlphaScreen™ assay. Table 63 further indicates the superiority ofV264I/I332E and I332E over S298A and S298A/E333A/K334A; whereasS298A/E333A/K334A improves Fc binding to V158 and F158 FcγRIIIa by1.7-fold and 4.7-fold respectively, I332E shows binding enhancements of2.2-fold and 10.1-fold respectively, and V264I/I332E shows bindingenhancements of 4.0-fold and 14-fold respectively. Also worth noting isthat the affinity of V264I/I332E for F158 FcγRIIIa (52 nM) is betterthan that of WT for the V158 allotype (68 nM), suggesting that this Fcvariant, as well as those with even greater improvements in binding, mayenable the clinical efficacy of antibodies for the low responsivepatient population to achieve that currently possible for highresponders. The correlation between the SPR and AlphaScreen™ bindingmeasurements are shown in FIGS. 23 a-23 d. FIGS. 23 a and 23 b show theKd-IC50 correlations for binding to V158 FcγRIIIa and F158 FcγRIIIarespectively, and FIGS. 23 c and 23 d show the fold-improvementcorrelations for binding to V158 FcγRIIIa and F158 FcγRIIIarespectively. The good fits of these data to straight lines (r²=0.9,r²=0.84, r²=0.98, and r²=0.90) support the accuracy the AlphaScreen™measurements, and validate its use for determining the relative FcγRbinding affinities of Fc variants.

SPR data were also acquired for binding of select trastuzumab Fcvariants to human V158 FcγRIIIa, F158 FcγRIIIa, and FcγRIIb. These dataare shown in Table 64. The Fc variants tested show substantial bindingenhancements to the activating receptor FcγRIIIa, with over 100-foldtighter binding observed for interaction of S239D/I332E/S298A with F158FcγRIIIa. Furthermore, for the best FcγRIIIa binders, F158FcγRIIIa/FcγRIIb ratios of 3-4 are observed. TABLE 64 SPR V158 SPR F158SPR FcγRIIIa FcγRIIIa FcγRIIb Kd Kd IC50 (nM) Fold (nM) Fold (nM) FoldWT 363.5 503 769 V264I/I332E 76.9 4.7 252 2.0 756 1.0 V264I/I332E/ 113.03.2 88 5.7 353 2.2 A330L S239D/I332E/ 8.2 44.3 8.9 56.5 46 16.7 A330LS239D/I332E/ 8.7 41.8 4.9 102.7 32 24.0 S298A S239D/I332E/ 12.7 28.6 6.379.8 35 22.0 V264I/A330L

Example 7 ADCC of Fc Variants

In order to determine the effect on effector function, cell-based ADCCassays were performed on select Fc variants. ADCC was measured using theDELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, Mass.) withpurified human peripheral blood monocytes (PBMCs) as effector cells.Target cells were loaded with BATDA at 1×10⁶ cells/ml, washed 4 timesand seeded into 96-well plate at 10,000 cells/well. The target cellswere then opsonized using Fc variant or WT antibodies at the indicatedfinal concentration. Human PBMCs, isolated from buffy-coat were added atthe indicated fold-excess of target cells and the plate was incubated at37° C. for 4 hrs. The co-cultured cells were centrifuged at 500×g,supernatants were transferred to a separate plate and incubated with Eusolution, and relative fluorescence units were measured using a PackardFusion™ α-FP HT reader (Packard Biosciences, IL). Samples were run intriplicate to provide error estimates (n=3, +/−S.D.). PBMCs wereallotyped for the V158 or F158 FcγRIIIa allotype using PCR.

ADCC assays were run on Fc variant and WT alemtuzumab using DoHH-2lymphoma target cells. FIG. 24 a is a bar graph showing the ADCC ofthese proteins at 10 ng/ml antibody. Results show that alemtuzumab Fcvariants I332E, V2641, and I332EN264I have substantially enhanced ADCCcompared to WT alemtuzumab, with the relative ADCC enhancementsproportional to their binding improvements to FcγRIIIa as indicated byAlphaScreen™ assay and SPR. The dose dependence of ADCC on antibodyconcentration is shown in FIG. 24 b. The binding data were normalized tothe minimum and maximum fluorescence signal for each particular curve,provided by the baselines at low and high antibody concentrationsrespectively. The data were fit to a sigmoidal dose-response model usingnonlinear regression, represented by the curve in the figure. The fitsenable determination of the effective concentration 50% (EC50) (i.e. theconcentration required for 50% effectiveness), which provides therelative enhancements to ADCC for each Fc variant. The EC50s for thesebinding data are analogous to the IC50s obtained from the AlphaScreen™competition data, and derivation of these values is thus analogous tothat described in Example 2 and FIG. 11. In FIG. 24 b, the log(EC50)s,obtained from the fits to the data, for WT, V264I/I332E, and S239D/I332Ealemtuzumab are 0.99, 0.60, and 0.49 respectively, and therefore theirrespective EC50s are 9.9, 4.0, and 3.0. Thus V264I/I332E and S239E/I332Eprovide a 2.5-fold and 3.3-fold enhancement respectively in ADCC over WTalemtuzumab using PBMCs expressing heterozygous V158/F158 FcγRIIIa.These data are summarized in Table 65 below. TABLE 65 Fold Improvementlog(EC50) EC50 (ng/ml) Over WT WT 0.99 9.9 V264I/I332E 0.60 4.0 2.5S239D/I332E 0.49 3.0 3.3

In order to determine whether these ADCC enhancements are broadlyapplicable to antibodies, select Fc variants were evaluated in thecontext of trastuzumab and rituximab. ADCC assays were run on Fc variantand WT trastuzumab using two breast carcinoma target cell lines BT474and Sk-Br-3. FIG. 25 a shows a bar graph illustrating ADCC at 1 ng/mlantibody. Results indicate that V264I and V264I/I332E trastuzumabprovide substantially enhanced ADCC compared to WT trastuzumab, with therelative ADCC enhancements proportional to their binding improvements toFcγRIIIa as indicated by AlphaScreen™ assay and SPR. FIGS. 25 b and 25 cshow the dose dependence of ADCC on antibody concentration for select Fcvariants. The EC50s obtained from the fits of these data and therelative fold-improvements in ADCC are provided in Table 66 below.Significant ADCC improvements are observed for I332E trastuzumab whencombined with A330L and A330Y. Furthermore, S239D/A330L/I332E provides asubstantial ADCC enhancement, greater than 300-fold for PBMCs expressinghomozygous F158/F158 FcγRIIIa, relative to WT trastuzumab andS298A/E333A/K334A, consistent with the FcγR binding data observed by theAlphaScreen™ assay and SPR. TABLE 66 Fold Improvement log(EC50) EC50(ng/ml) Over WT FIG. 25b WT 1.1 11.5 I332E 0.34 2.2 5.2 A330Y/I332E−0.04 0.9 12.8 A330L/I332E 0.04 1.1 10.5 FIG. 25d WT −0.15 0.71S298A/E333A/K334A −0.72 0.20 3.6 S239D/A330L/I332E −2.65 0.0022 323

ADCC assays were run on V264I/I332E, WT, and S298A/D333A/K334A rituximabusing WIL2-S lymphoma target cells. FIG. 26 a presents a bar graphshowing the ADCC of these proteins at 1 ng/ml antibody. Results indicatethat V264I/I332E rituximab provides substantially enhanced ADCC relativeto WT rituximab, as well as superior ADCC to S298A/D333A/K334A,consistent with the FcγRIIIa binding improvements observed byAlphaScreen™ assay and SPR. FIGS. 26 b and 26 c show the dose dependenceof ADCC on antibody concentration for select Fc variants. The EC50sobtained from the fits of these data and the relative fold-improvementsin ADCC are provided in Table 67 below. As can be seen S239D/I332E/A330Lrituximab provides greater than 900-fold enhancement in EC50 over WT forPBMCs expressing homozygous F158/F158 FcγRIIIa. The differences in ADCCenhancements observed for alemtuzumab, trastuzumab, and rituximab arelikely due to the use of different PBMCs, different antibodies, anddifferent target cell lines. TABLE 67 Fold Improvement log(EC50) EC50(ng/ml) Over WT FIG. 26b WT 0.23 1.7 S298A/E333A/K334A −0.44 0.37 4.6V264I/I332E −0.83 0.15 11.3 FIG. 26c WT 0.77 5.9 S239D/I332E/A330L −2.200.0063 937

Thus far, ADCC data has been normalized such that the lower and upperbaselines of each Fc polypeptide are set to the minimal and maximalfluorescence signal for that specific Fc polypeptide, typically beingthe fluorescence signal at the lowest and highest antibodyconcentrations respectively. Although presenting the data in this matterenables a straightforward visual comparison of the relative EC50s ofdifferent antibodies (hence the reason for presenting them in this way),important information regarding the absolute level of effector functionachieved by each Fc polypeptide is lost. FIGS. 27 a and 27 b presentcell-based ADCC data for trastuzumab and rituximab respectively thathave been normalized according to the absolute minimal lysis for theassay, provided by the fluorescence signal of target cells in thepresence of PBMCs alone (no antibody), and the absolute maximal lysisfor the assay, provided by the fluorescence signal of target cells inthe presence of Triton X1000. The graphs show that the antibodies differnot only in their EC50, reflecting their relative potency, but also inthe maximal level of ADCC attainable by the antibodies at saturatingconcentrations, reflecting their relative efficacy. Thus far these twoterms, potency and efficacy, have been used loosely to refer to desiredclinical properties. In the current experimental context, however, theyare denoted as specific quantities, and therefore are here explicitlydefined. By “Potency” as used in the current experimental context ismeant the EC50 of an antibody or Fc fusion. By “efficacy” as used in thecurrent experimental context is meant the maximal possible effectorfunction of an antibody or Fc fusion at saturating levels. In additionto the substantial enhancements to potency described thus far, FIGS. 27a and 27 b show that the Fc variants of the present invention providegreater than 100% enhancements in efficacy over WT trastuzumab andrituximab.

A critical parameter governing the clinical efficacy of anti-cancerantibodies is the expression level of target antigen on the surface oftumor cells. Thus a major clinical advantage of Fc variants that enhanceADCC may be that it enables the targeting of tumors that express lowerlevels of antigen. In To test this hypothesis, WT and Fc varianttrastuzumab antibodies were tested for their ability to mediate ADCCagainst different cell lines expressing varying levels of the Her2/neutarget antigen. ADCC assays were run with various cell lines expressingamplified to low levels of Her2/neu receptor, including Sk-Br-3 (1×10⁶copies), SkOV3 (˜1×10⁵), OVCAR3(˜1×10⁴), and MCF-7 (˜3×10³ copies),using the DELFIA EuTDA Cytotoxicity kit (PerkinElmer, Boston, Mass.).Target cells were loaded with BATDA in batch for 25 minutes, washedmultiple times with medium and seeded at 10,000 cells per well in96-well plates. Target cells were opsonized for 15 minutes with variousantibodies and concentrations (final conc. ranging from 100 ng/ml to0.0316 ng/ml in ½ log steps, including no treatment control). HumanPBMCs, isolated from buffy-coat and allotyped as homozygous F158/F158FcγRIIIa were then added to opsonized cells at 25-fold excess andco-cultured at 37° C. for 4 hrs. Thereafter, plates were centrifuged,supernatants were removed and treated with Eu3+ solution, and relativefluorescence units (correlating to the level of cell lysis) weremeasured using a Packard Fusion™ α-FP HT reader (PerkinElmer, Boston,Mass.). The experiment was carried out in triplicates. FIG. 28 shows theADCC data comparing WT and Fc variant trastuzumab against the fourdifferent Her2/neu⁺ cell lines. The S239D/I332E and S239D/I332E/A330Lvariants provide substantial ADCC enhancements over WT trastuzumab athigh, moderate, and low expression levels of target antigen. This resultsuggests that the Fc variants of the present invention may broaden thetherapeutic window of anti-cancer antibodies.

Natural killer (NK) cells are a subpopulation of cells present in PBMCsthat are thought to play a significant role in ADCC. Select Fc variantswere tested in a cell-based ADCC assay in which natural killer (NK)cells rather than PBMCs were used as effector cells. In this assay therelease of endogenous lactose dehydrogenase (LDH), rather than EuTDA,was used to monitor cell lysis. FIG. 29 shows that the Fc variants showsubstantial ADCC enhancement when NK cells are used as effector cells.Furthermore, together with previous assays, the results indicate thatthe Fc variants of the present invention show substantial ADCCenhancements regardless of the type of effector cell or the detectionmethod used.

Example 8 ADCP of Fc Variants

Another important FcγR-mediated effector function is ADCP. Phagocytosisof target cancer cells may not only lead to the immediate destruction oftarget cells, but because phagocytosis is a potential mechanism forantigen uptake and processing by antigen presenting cells, enhanced ADCPmay also improve the capacity of the antibody or Fc fusion to elicit anadaptive immune response. The ability of the Fc variants of the presentinvention to mediate ADCP was therefore investigated. Monocytes wereisolated from heterozygous V158/F158 FcγRIIIa PBMCs using a Percollgradient. After one week in culture in the presence of 0.1 ng/ml,differentiated macrophages were detached with EDTA/PBS- and labeled withthe lipophilic fluorophore, PKH26, according to the manufacturer'sprotocol (Sigma, St Louis, Mo.). Sk-Br-3 target cells were labeled withPKH67 (Sigma, St Louis, Mo.), seeded in a 96-well plate at 20,000 cellsper well, and treated with designated final concentrations of WT or Fcvariant trastuzumab. PKH26-labeled macrophages were then added to theopsonized, labeled Sk-Br-3 cells at 20,000 cells per well and the cellswere co-cultured for 18 hrs before processing cells for analysis of duallabel flow cytometry. Percent phagocytosis was determined as the numberof cells co-labeled with PKH76 and PKH26 (macrophage+Sk-Br-3) over thetotal number of Sk-Br-3 in the population(phagocytosed+non-phagocytosed) after 10,000 counts. FIG. 30 shows datacomparing WT and Fc variant trastuzumab at various antibodyconcentrations. The results indicate that the S239D/I332E/A330L variantprovides a significant enhancement in ADCP over WT trastuzumab.

Example 9 Complement Binding and Activation by Fc Variants

Complement protein C1q binds to a site on Fc that is proximal to theFcγR binding site, and therefore it was prudent to determine whether theFc variants have maintained their capacity to recruit and activatecomplement. The AlphaScreen™ assay was used to measure binding of selectFc variants to the complement protein C1q. The assay was carried outwith biotinylated WT alemtuzumab antibody attached to streptavidin donorbeads as described in Example 2, and using C1q coupled directly toacceptor beads. Binding data of V264I,I332E, S239E, and V264I/I332Erituximab shown in FIG. 31 a indicate that C1q binding is uncompromised.Cell-based CDC assays were also performed on select Fc variants toinvestigate whether Fc variants maintain the capacity to activatecomplement. Alamar Blue was used to monitor lysis of Fc variant and WTrituximab-opsonized WIL2-S lymphoma cells by human serum complement(Quidel, San Diego, Calif.). The data in FIG. 31 b show that CDC isuncompromised for the Fc variants S239E, V264I, and V264I/I332Erituximab. In contrast, FIG. 31 c shows that CDC of the Fc variantS239D/I332E/A330L is completely ablated, whereas the S239D/I332E variantmediates CDC that is comparable to WT rituximab. These results indicatethat protein engineering can be used to distinguish between differenteffector functions. Such control will not only enable the generation ofantibodies and Fc fusions with properties tailored for a desiredclinical outcome, but also provide a unique set of reagents with whichto experimentally investigate effector function biology.

Example 10 Protein A and FcRn Binding by Fc Variants

As discussed, bacterial proteins A and G and the neonatal Fc receptorFcRn bind to the Fc region between the Cγ2 and Cγ3 domains. Protein A isfrequently employed for antibody purification, and FcRn plays a key rolein antibody pharmacokinetics and transport. It was therefore importantto investigate the ability of the Fc variants of the present inventionto bind protein A and FcRn. The AlphaScreen™ assay was used to measurebinding of select Fc variants to protein A and human FcRn usingbiotinylated WT alemtuzumab antibody attached to streptavidin donorbeads as described in Example 2, and using protein A and FcRn coupleddirectly to acceptor beads. The binding data are shown in FIG. 32 forprotein A and FIG. 33 for FcRn. The results indicate that the Cγ2-Cγ3hinge region is unaffected by the Fc substitutions, and importantly thatthe capacity of the Fc variants to bind protein A and FcRn isuncompromised.

Example 11 Capacity of Fc Variants to Bind Mouse FcγRs

Optimization of Fc to nonhuman FcγRs may be useful for experimentallytesting Fc variants in animal models. For example, when tested in mice(for example nude mice, SCID mice, xenograft mice, and/or transgenicmice), antibodies and Fc fusions that comprise Fc variants that areoptimized for one or more mouse FcγRs may provide valuable informationwith regard to clinical efficacy, mechanism of action, and the like. Inorder to evaluate whether the Fc variants of the present invention maybe useful in such experiments, affinity of select Fc variants for mouseFcγRIII was measured using the AlphaScreen™ assay. The AlphaScreen™assay was carried out using biotinylated WT alemtuzumab attached tostreptavidin donor beads as described in Example 2, and GST-tagged mouseFcγRIII bound to glutathione chelate acceptor beads, expressed andpurified as described in Example 2. These binding data are shown inFIGS. 34 a and 34 b in the context of alemtuzumab and trastuzumabrespectively. Results show that some Fc variants that enhance binding tohuman FcγRIIIa also enhance binding to mouse FcγRIII. The enhancement ofmouse effector function by the Fc variants was investigated byperforming the aforementioned cell-based ADCC assays using mouse ratherthan human PBMC's. FIG. 35 shows that the S239D/I332E/A330L trastuzumabvariant provides substantial ADCC enhancement over WT in the presence ofmouse immune cells. This result indicates that the Fc variants of thepresent invention, or other Fc variants that are optimized for nonhumanFcγRs, may find use in experiments that use animal models.

Example 12 Validation of Fc Variants Expressed in CHO Cells

Whereas the Fc variants of the present invention were expressed in 293Tcells for screening purposes, large scale production of antibodies istypically carried out by expression in Chinese Hamster Ovary (CHO) celllines. In order to evaluate the properties of CHO-expressed Fc variants,select Fc variants and WT alemtuzumab were expressed in CHO cells andpurified as described in Example 2. FIG. 36 shows AlphaScreen™ datacomparing binding of CHO- and 293T-expressed Fc variant and WTalemtuzumab to human V158 FcγRIIIa. The results indicate that the Fcvariants of the present invention show comparable FcγR bindingenhancements whether expressed in 293T or CHO.

Example 13 Enhancement of Fc Variants in Fucose Minus Strain

Combinations of the Fc variants of the present invention with other Fcmodifications are contemplated with the goal of generating novelantibodies or Fc fusions with optimized properties. It may be beneficialto combine the Fc variants of the present invention with other Fcmodifications, including modifications that alter effector function orinteraction with one or more Fc ligands. Such combination may provideadditive, synergistic, or novel properties in antibodies or Fc fusions.For example, a number of methods exist for engineering differentglycoforms of Fc that alter effector function. Engineered glycoforms maybe generated by a variety of methods known in the art, many of thesetechniques are based on controlling the level of fucosylated and/orbisecting oligosaccharides that are covalently attached to the Fcregion. One method for engineering Fc glycoforms is to express the Fcpolypeptide in a cell line that generates altered glycoforms, forexample Lec-13 CHO cells. In order to investigate the properties of Fcvariants combined with engineered glycoforms, WT and V209(S239D/I332E/A330L) trastuzumab were expressed in Lec-13 CHO cells andpurified as described above. FIG. 37 a shows AlphaScreen™ binding datacomparing the binding to human V158 FcγRIIIa by WT and V209 trastuzumabexpressed in 293T, CHO, and Lec-13 cells. The results show that there issubstantial synergy between the engineered glycoforms produced by thiscell line and the Fc variants of the present invention. The cell-basedADCC assay, shown in FIG. 37 b, supports this result. Together thesedata indicate that other Fc modifications, particularly engineeredglycoforms, may be combined with the Fc variants of the presentinvention to generate antibodies and Fc fusions with optimized effectorfunctions.

Example 14 Therapeutic Application of Fc Variants

A number of Fc variants described in the present invention havesignificant potential for improving the therapeutic efficacy ofanticancer antibodies. For illustration purposes, a number of Fcvariants of the present invention have been incorporated into thesequence of the antibody rituximab. The WT rituximab light chain andheavy chain, described in U.S. Pat. No. 5,736,137, are provided in FIGS.38 a and 38 b. The improved anti-CD20 antibody sequences are provided inFIG. 38 c. The improved anti-CD20 antibody sequences comprise at leastnon-WT amino acid selected from the group consisting of X₁, X₂, X₃, X₄,X₅, X₆, X₇, and X₈. These improved anti-CD20 antibody sequences may alsocomprise a substitution Z₁ and/or Z₂. The use of rituximab here issolely an example, and is not meant to constrain application of the Fcvariants to this antibody or any other particular antibody or Fc fusion.

Example 15 A complete Structure/Function Analysis Fc/Fc LigandSpecificity

It is clear from the results of these experiments that proteinengineering is a powerful tool for mining Fc substitutions thatsignificantly alter its biological function and specificity. Given theprofound clinical value of antibodies and Fc fusions, the implication isthat the protein engineering methods of the present invention can beused to tune the clinical properties of these important biotherapeutics.Such capability, however, demands a more complete understanding of therelationship between the structure and function of Fc and Fc ligands. Inaddition, the lack of available information on the determinants of Fc/Fcligand specificity means that it is not possible to actively design Fcvariants with all desired properties as target goals. Thus it is likelythat, despite the aggressive experimental effort described in thepresent invention, there are therapeutically useful Fc variants thathave not been mined, and biochemical properties of Fc variants thatremain undiscovered. Equally important to obtaining new Fc variants forbiotherapeutic application is the ability to improve the predictivenessof the design method, thereby permitting variants to be identified evenmore efficiently. Towards these goals, a more thorough characterizationof Fc/Fc ligand biology was carried out. This included: 1) an expansionof the primary screen to include all relevant Fc ligands, and 2) anincrease in the number of Fc variants to explore a greater set ofsubstitutions at all relevant Fc positions. Together this broadenedapproach will enable a more thorough mining of useful Fc variants,provide a greater understanding of Fc/Fc ligand specificity and biology,and provide a greater data set to enable a rigorous quantitativeassessment of the predictiveness of the design methods.

Expansion of the Primary Screen

In order to better characterize the structural and functionaldeterminants of Fc specificity, the primary screen was expanded toinclude all relevant Fc ligands. Thus all Fc variants are tested inparrallel for binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa(Val158 isoform), FcRn, and C1q. The AlphaScreen™ assay was used asdescribed above. All Fc variants were screened in the context of eitheralemtuzumab or trastuzumab according to Table 62. Table 68 shows anexample of the parrallel screen for a set of substitutions at Fcpositions 234 and 235. In this table, light grey indicates that Fcvariant/Fc ligand affinity is 0.5-fold or less than WT, medium greyindicates that Fc variant/Fc ligand affinity is within 0.5-2.0 of WT,dark grey indicates that Fc variant/Fc ligand affinity is increased by2-fold or greater, and white indicates that the Fc variant/Fc ligandinteraction was not measured or that the data did not allow an accuratedetermination of affinity. Thus Fc variants are grouped as those thatsignificantly decrease, those that do not substantially alter, and thosethat significantly increase binding to a given Fc ligand. Visualizationof the data in this way provides a structure/function map of Fc,enabling a straightforward interpretation of the results for eachposition such that useful and interesting variants can be efficientlyidentified, and such that predictiveness of the design method can beassessed in a practical manner. TABLE 68

A number of substitutions at positions 234 and 235 show differenctspecificities for binding to the various Fc ligands. Although thedifferences in some cases are subtle, the results indicate that it isindeed possible to engineer Fc specificity for different Fc ligands,even at at the FcγR interface where a number of highly homologousreceptors bind to the same site. Other Fc variants that provide moredistinct affinity differences are presented in Table 69. TABLE 69

These data show even more convincingly that it is possible to tune Fcfor Fc ligand specificity, often by using very subtle mutationaldifferences. For example, the A330Y/I332E variant enhances binding toall FcγRs, particularly FcγRIIIa, as well as FcRn, while maintainingbinding to C1q. However the A300L/I332E variant shows enhanced bindingto FcγRI and FcγRIIIa, but has WT affinity for the FcγRII's. Incontrast, mutations at L328 provide preferential enhancement of theFcγRII's over FcγRI and FcγRIIIa. In the case of the L328E/I332Evariant, affinity for all FcγRII's is increased, whereas L328T/I332Eprovides a clear enhancement specificity profile ofFcγRIIc>FcγRIIb>FcγRIIa. In contrast, L328A significantly enhancesbinding to FcγRIIa, but provides WT affinity for all other FcγR'sincluding FcγRIIb and FcγRIIc. It is clear from these results that verysubtle mutational differences can provide substantial differences inspecificity. Accordingly, collections of Fc variants such as these willnot only enable the generation of antibodies and Fc fusions that haveeffector function tailored for the desired outcome, but they alsoprovide a unique set of reagents with which to experimentallyinvestigate and characterize effector function biology.

Expansion of the Fc Variant Set

Because of the incomplete information concerning the structural andfunctional determinants of Fc/Fc ligand interaction, it has not beenpossible to actively engineer Fc for all desired optimization goals. Thedistinct specificity differences observed in Tables 68 and 69 to thevarious FcγRs were due more to the aggressive screening approach of thepresent invention; these Fc variants were not actively designed withtheir particular properties as the target goals due to the lack ofstructural information for binding of Fc to the different FcγRs, as wellas the lack of understanding of how the structure and flexibility of thehinge impacts FcγR binding. Indeed the decision to explore a largenumber and variety of substitutions at these positions 234 and 235 wasbased on the knowledge that they are near the Fc/FcγR binding site, thatmutations at these positions affect FcγR binding, and that according tocomputational screening calculations a large number and variety ofsubstitutions are permissible at these positions. Overall, the lack ofstructural information on the determinants of Fc/FcγR specificity, thelack of high-resolution structural information for the Fc/C1q complex,and the inability to account for indirect affects of substitutions onFc/Fc ligand binding, together make it a certainty that all of theinteresting and potentially useful Fc variants will not be exploredusing the current engineering methods. In order to fully mine useful Fcvariants, as well as to obtain a more complete picture of the structuraland function determinants of Fc/Fc ligand interaction, the set of Fcvariants was expanded to explore a broader set of mutations. All Fcpositions at or near the binding sites for FcγR's and C1q, chosen byvisual inspection of the available structures and using the informationprovided by the results of previous Fc variant screening, were saturatedsuch that all substitutions were constructed that have not been testedpreviously. At Fc positions significantly distal to the FcγR and C1qbinding sites, a subset of select substitutions were designed based onpredicted energies in previously described computational screeningcalculations, and based on available data from existing Fc variants.This new set of Fc variants, 576 total, is presented in Table 70. TABLE70 Position WT Substitution(s) Variant 221 D KY 801-802 222 K EY 513-514223 T EK 803-804 224 H EY 805-806 225 T EKW 807-809 227 P EKYG 705-708228 P EKYG 709-712 230 P EYG 609-611 231 A EKYPG 612-616 232 P EKYG321-324 233 E NQKRSTHAVLIFMYWG 617-632 234 L KRSAMWPG 417-424 235 LEKRAMWPG 425-432 236 G DENQKRSTHAVLIFMYWP 713-730 237 GDENQKRSTHVLIFMYWP 731-747 238 P DENQKRSTHVLIFMYWG 748-764 239 SQKRVLIMWPG 325-334 241 F DEY 335-337 243 F E 515 246 K DEHY 810-813 249D QHY 814-816 255 R EY 817-818 258 E SHY 819-821 260 T DEHY 822-825 262V EF 826-827 264 V DENQKRSHWPG 433-443 265 D QKRSTHVLIFMYWP 444-457 267S EQKRVLIFMYWP 338-349 268 H DEQKRTVLIFMWPG 350-363 269 E KSVIMWPG765-772 270 D RSLIFMYWPG 516-525 271 P DENQKRSTHAVLIFMYWG 526-543 272 EDRTHVLFMWPG 633-643 274 K DNSHVIFMWPG 644-654 275 F L 828 276 NDTHVIFMWPG 655-664 278 Y DNQRSHVLIMPG 665-676 280 D KLWPG 544-548 281 GDKYP 829-832 282 V EKYPG 833-837 283 E KHLYPG 838-843 284 V ENTLY844-848 285 H DEQKYW 773-778 286 N EYPG 779-782 288 K DEY 783-785 290 KDNHLW 549-553 291 P DEQTHIG 849-855 292 R DETY 786-789 293 ENRSTHVLIFMYWPG 554-567 294 E KRSTHVLIFMYWPG 568-581 295 QDENRSTHVIFMYWPG 582-596 296 Y KRAVMG 597-602  297* N QKRTHVLIFMYWPG856-869 298 S DEQKRIFMYW 364-373 299 T DENQKRLFMYWPG 374-386 300 YDENQKRSTHAVMWPG 387-401 301 R DEHY 870-873 303 V DEY 874-876 304 S DNTHL877-881 305 V ETY 882-884 317 K EQ 885-886 318 E QHLY 887-890 320 KNSHVLFYWPG 677-686 322 K DSVIFYWPG 687-695 324 S HFMWPG 603-608 325 NKRSFMYWPG 696-704 326 K P 458 327 A EKRHVIFMYWP 459-469 328 LDQKRSTVIYWPG 470-481 329 P DENQKRSTHVLIMYWG 482-497 330 A ENTPG 402-406331 P DQRTLIFMYW 498-507 332 I KRSVLFMWPG 407-416 333 E LFMP 508-511 334K P 512 335 T NSHVLIFMWPG 790-800 336 I EKY 891-893 337 S ENH 894-896* Substitutions at 297 were made in the context of S239D/I332E

All references are herein expressly incorporated by reference.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

1. A variant protein comprising an Fc variant of a wild-type Fcpolypeptide, said Fc variant comprising at least one amino acidmodification in the Fc region of said wild-type Fc polypeptide, whereinsaid variant protein exhibits altered binding to an FcγR as compared tothe wild-type Fc polypeptide.
 2. A variant protein according to claim 1,wherein said Fc variant comprises at least one substitution at aposition corresponding to an Fc position selected from the groupconsisting of: 230, 240, 244, 245, 247, 262, 263, 266, 273, 275, 299,302, 313, 323, 325, 328, and
 332. 3. A variant protein according toclaim 1 wherein said altered binding is an increase in affinity of saidFc variant to said FcγR.
 4. A variant protein according to claim 3,wherein said Fc variant binds with greater affinity to a mouse FcγR. 5.A variant protein according to claim 3, wherein said said FcγR is ahuman Fc receptor selected from the group consisting of FcγRI, FcγRIIa,FcγRIIb, FcγRIIc, and FcγRIIIa.
 6. A variant protein according to claim3, wherein said Fc variant binds with greater affinity to human FcγRIand FcγRIIIa, but exhibits unaltered affinity to a human receptorselected from the group consisting of FcγRIIa, FcγRIIb, and FcγRIIc. 7.A variant protein according to claim 6, wherein said Fc variant exhibitsunaltered affinity to FcγRIIa, FcγRIIb, and FcγRIIc.
 8. A variantprotein according to claim 3, wherein the affinity increase for bindingto one or more human FcγRII's is greater than the affinity increase forbinding to human FcγRI and FγRIIIa.
 9. A variant protein according toclaim 3, wherein the affinity increase is the same for binding toFcγRIIa, FcγRIIb, and FcγRIIc.
 10. A variant protein according to claim9, wherein the affinity increase for binding to FcγRIIc is greater thanthe affinity increase for binding to FcγRIIb.
 11. A variant proteinaccording to claim 3, wherein said Fc variant binds with greateraffinity to human FcγRIIa, but exhibits unaltered affinity to a humanreceptor selected from the group consisting of FcγRI, FcγRIIb, FcγRIIc,and FγRIIa.
 12. A variant protein according to claim 11, wherein said Fcvariant exhibits unaltered affinity to FcγRI, FcγRIIb, FcγRIIc, andFγRIIIa.
 13. A variant protein according to any of claims 1-12, whereinsaid Fc variant binds with greater affinity to FcRn.
 14. A variantprotein according to any of claims 1-12, wherein said Fc variant bindswith unaltered affinity to FcRn.
 15. A variant protein according to anyof claims 1-12, wherein said Fc variant binds with reduced affinity toFcRn.
 16. A variant protein according to any of claims 1-12, whereinsaid Fc variant binds with greater affinity to C1q.
 17. A variantprotein according to any of claims 1-12, wherein said Fc variant bindswith unaltered affinity to C1q.
 18. A variant protein according to claim1 wherein said Fc variant further comprises an engineered glycoform. 19.A variant protein according to claim 18 wherein said engineeredglycoform comprises an altered level of fucosylation or bisectingoligosaccharides as compared to the parent Fc polypeptide.
 20. A variantprotein according to claim 18 wherein said engineered glycoform improveseffector function.
 21. A variant protein comprising an Fc variant of awild-type Fc polypeptide, said Fc variant comprising at least one aminoacid modification in the Fc region of said wild-type Fc polypeptide,wherein said Fc variant modulates effector function as compared to theparent Fc polypeptide.
 22. A variant protein according to claim 21,wherein said effector function is ADCC.
 23. A variant protein accordingto claim 22, wherein said Fc variant improves ADCC in the presence ofhuman effector cells as compared to said wild-type Fc polypeptide.
 24. Avariant protein according to claim 2 wherein said ADCC improvement is anenhancement in potency such that the EC50 of said Fc variant isapproximately 5-fold greater than that of said parent Fc polypeptide.25. A variant protein according to claim 23 wherein said ADCCimprovement is an enhancement in potency such that the EC50 of said Fcvariant is between approximately 5-fold and 1000-fold greater than thatof said parent Fc polypeptide.
 26. A variant protein according to claim23 wherein said ADCC improvement is an enhancement in efficacy such thatthe maximal ADCC is approximately 2-fold greater than that of saidparent Fc polypeptide.
 27. A variant protein according to claim 26wherein said Fc variant improves ADCC in the presence of mouse effectorcells as compared to said wild-type Fc polypeptide.
 28. A variantprotein according to claim 21 wherein said effector function is ADCP.29. A variant protein according to claim 28 wherein said Fc variantimproves ADCP as compared to said wild-type Fc polypeptide.
 30. Avariant protein according to claim 25 or 29 wherein CDC is unaffected.31. A variant protein according to claim 25 or 29 wherein CDC isablated.
 32. A variant protein according to claim 1 that has specificityfor a target antigen selected from the group consisting of CD20, CD22,CD33, CD52, Her2/neu, EGFR, EpCAM, MUC1, GD3, CEA, CA 125, HLA-DR,TNFalpha, and VEGF.
 33. A variant protein according to claim 1 or 21comprising a modification selected from the group consisting of 239D,239E, 239N, 239Q, 239T, 240I, 240M, 264I, 264T, 264Y, 297D, 330I, 330L,330Y, 332D, 332E, 332N, 332Q, A231E, A231G, A231K, A231P, A231Y, A298H,A327D, A327E, A327F, A327H, A327I, A327K, A327L, A327M, A327N, A327P,A327R, A327T, A327T, A327V, A327W, A327Y, A330E, A330F, A330F, A330G,A330H, A330I, A330L, A330L/I332E, A330M, A330N, A330P, A330R, A330S,A330T, A330V, A330W, A330Y, A330Y, A330Y/I332E, D221K, D221Y, D249H,D249Q, D249Y, D265F, D265F/N297E/I332E, D265G, D265H, D265I, D265K,D265L, D265M, D265N, D265P, D265Q, D265R, D265S, D265T, D265V, D265W,D265Y, D265Y/N297D/I332E, D265Y/N297D/T299L/I332E, D270F, D270G, D270H,D270I, D270L, D270M, D270P, D270Q, D270R, D270S, D270T, D270W, D270Y,D280G, D280H, D280K, D280L, D280P, D280Q, D280W, D280Y, E233A, E233D,E233F, E233G, E233H, E233I, E233K, E233L, E233M, E233N, E233Q, E233R,E233S, E233T, E233V, E233W, E233Y, E258H, E258S, E258Y, E269F, E269G,E269H, E269I, E269K, E269L, E269M, E269N, E269P, E269R, E269S, E269T,E269V, E269W, E269Y, E272D, E272F, E272G, E272H, E272I, E272K, E272L,E272M, E272P, E272R, E272S, E272T, E272V, E272W, E272Y, E283G, E283H,E283K, E283L, E283P, E283R, E283Y, E293F, E293G, E293H, E293I, E293L,E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W, E293Y, E294F,E294G, E294H, E294I, E294K, E294L, E294M, E294N, E294P, E294R, E294S,E294T, E294V, E294W, E294Y, E318H, E318L, E318Q, E318R, E318Y, E333A,E333F, E333H, E333I, E333L, E333M, E333P, E333S, E333T, E333Y, E333Y,F241D, F241E, F241E/F243Q/V262T/V264E, F241 E/F243Q/V262T/V264E/I332E,F241 E/F243R/V262E/V264R, F241 E/F243R/V262E/V264R/I332E, F241E/F243Y/V262T/V264R, F241E/F243Y/V262T/V264R/I332E, F241L,F241L/F243L/V262I/V264I, F241L/V262I, F241R, F241R/F243Q/V262T/V264R,F241R/F243Q/V262T/V264R/I332E, F241W, F241W/F243W,F241W/F243W/V262A/V264A, F241Y, F241Y/F243Y/V262T/V264T,F241Y/F243Y/V262T/V264T/N297D/I332E, F243E, F243L, F243L/V262I/V264W,F243L/V264I, F243Q, F243R, F243W, F243W, F243Y, F275L, F275W, F275W,G236A, G236D, G236E, G236F, G236H, G236I, G236K, G236L, G236M, G236N,G236P, G236Q, G236R, G236S, G236T, G236V, G236W, G236Y, G237D, G237E,G237F, G237H, G237I, G237K, G237L, G237M, G237N, G237P, G237Q, G237R,G237S, G237T, G237V, G237W, G237Y, G281D, G281K, G281P, G281Y, H224E,H224Y, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268M, H268P,H268Q, H268R, H268T, H268V, H268W, H285D, H285E, H285K, H285Q, H285W,H285Y, I332A, I332D, I332E, I332F, I332G, I332H, I332K, I332L, I332M,I332N, I332N, I332P, I332Q, I332R, I332S, I332T, I332V, I332W, I332Y,I332Y, I336E, I336K, I336Y, K222E, K222Y, K246D, K246E, K246H, K246Y,K274D, K274E, K274F, K274G, K274H, K274I, K274L, K274M, K274N, K274P,K274R, K274S, K274T, K274V, K274W, K274Y, K288D, K288E, K288Y, K290D,K290G, K290H, K290L, K290N, K290S, K290T, K290W, K290Y, K317E, K317Q,K320D, K320F, K320G, K320H, K320I, K320L, K320N, K320P, K320S, K320T,K320T, K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K322I, K322P,K322S, K322T, K322V, K322W, K322Y, K325E, K326A, K326D, K326I, K326I,K326L, K326N, K326P, K326Q, K326S, K326T, K326T, K326W, K326Y, K334A,K334E, K334F, K334I, K334P, K334T, L234A, L234D, L234E, L234F, L234G,L234H, L234I, L234I, L234I/L235D, L234K, L234M, L234N, L234P, L234Q,L234R, L234S, L234T, L234V, L234W, L234Y, L235A, L235D, L235E, L235F,L235F, L235G, L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R,L235S, L235T, L235V, L235W, L235Y, L328A, L328D, L328D/I332E, L328E,L328E/I332E, L328F, L328G, L328H, L328H/I332E, L328I, L328I/I332E,L328K, L328M, L328M/I332E, L328N, L328N/I332E, L328P, L328Q, L328Q,L328Q/I332E, L328R, L328S, L328T, L328T/I332E, L328V, L328V/I332E,L328W, L328Y, M428F M428L, N276D, N276E, N276F, N276G, N276H, N276I,N276L, N276M, N276P, N276R, N276S, N276T, N276V, N276W, N276Y, N276Y,N286E, N286G, N286P, N286Y, N297D, N297D/A330Y/I332E, N297D/I332E,N297D/I332E/S239D/A330L, N297D/S298A/A330Y/I332E, N297D/T299E/I332E,N297D/T299F/I332E, N297D/T299H/I332E, N297D/T299I/I332E,N297D/T299L/I332E, N297D/T299V/I332E, N297E, N297E/I332E, N297F, N297G,N297H, N297I, N297K, N297L, N297M, N297P, N297Q, N297R, N297S, N297S,N297S/I332E, N297T, N297V, N297W, N297Y, N325A, N325D, N325E, N325F,N325G, N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N325Q, N325R,N325S, N325T, N325V, N325W, N325Y, P227E, P227G, P227K, P227Y, P228E,P228G, P228K, P228Y, P230A, P230A/E233D, P230A/E233D/I332E, P230E,P230G, P230Y, P232E, P232G, P232K, P232Y, P238D, P238E, P238F, P238G,P238H, P238I, P238K, P238L, P238M, P238N, P238Q, P238R, P238S, P238T,P238V, P238W, P238Y, P244H, P244H/P245A/P247V, P245A, P247G, P247V,P271A, P271D, P271E, P271 F, P271G, P271 H, P271I, P271K, P271L, P271M,P271N, P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, P291D, P291E,P291G, P291H, P291I, P291Q, P291T, P329D, P329E, P329F, P329F, P329G,P329H, P329I, P329K, P329L, P329M, P329N, P329Q, P329R, P329S, P329T,P329V, P329W, P329Y, P331D, P331F, P331H, P331H, P331I, P331L, P331M,P331Q, P331R, P331T, P331V, P331W, P331Y, Q295D, Q295E, Q295F, Q295G,Q295H, Q295I, Q295K, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V,Q295W, Q295Y, R255E, R255Y, R292D, R292E, R292T, R292Y, R301D, R301E,R301H, R301Y, S239D, S239D/A330L/I332E, S239D/A330Y/I332E,S239D/A330Y/I332E/K326E, S239D/A330Y/I332E/K326T,S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/L235D,S239D/A330Y/I332E/V240I, S239D/A330Y/I332E/V264T,S239D/A330Y/I332E/V266I, S239D/D265F/N297D/I332E,S239D/D265H/N297D/I332E, S239D/D265I/N297D/I332E,S239D/D265L/N297D/I332E, S239D/D265T/N297D/I332E,S239D/D265V/N297D/I332E, S239D/D265Y/N297D/I332E, S239D/I332D,S239D/I332E, S239D/I332E/A330I, S239D/I332N, S239D/I332Q,S239D/N297D/I332E, S239D/N297D/I332E/A330Y,S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T,S239D/N297D/I332E/K326E, S239D/N297D/I332E/L235D, S239D/S298A/I332E,S239D/V264I/A330L/I332E, S239D/V264I/I332E, S239D/V264I/S298A/I332E,S239E, S239E/D265G, S239E/D265N, S239E/D265Q, S239E/I332D, S239E/I332E,S239E/I332N, S239E/I332Q, S239E/N297D/I332E, S239E/V264I/A330Y/I332E,S239E/V264I/I332E, S239E/V264I/S298A/A330Y/I332E, S239F, S239G, S239H,S239I, S239K, S239L, S239M, S239N, S239N/A330L/I332E, S239N/A330Y/I332E,S239N/I332D, S239N/I332E, S239N/I332N, S239N/I332Q, S239N/S298A/I332E,S239P, S239Q, S239Q/I332D, S239Q/I332E, S239Q/I332N, S239Q/I332Q,S239Q/V264I/I332E, S239R, S239T, S239V, S239W, S239Y, S267D, S267E,S267F, S267H, S267I, S267K, S267L, S267L/A327S, S267M, S267N, S267P,S267Q, S267Q/A327S, S267R, S267T, S267V, S267W, S267Y, S298A,S298A/I332E, S298D, S298E, S298F, S298I, S298K, S298M, S298N, S298Q,S298R, S298V, S298W, S298Y, S304D, S304H, S304L, S304N, S304T, S324D,S324F, S324G, S324H, S324I, S324L, S324M, S324P, S324R, S324T, S324V,S324W, S324Y, S337E, S337H, S337N, T223E, T223K, T225, T225E, T225K,T225W, T250E, T250Q, T260D, T260E, T260H, T260Y, T299A, T299D, T299E,T299F, T299G, T299H, T299I, T299K, T299L, T299L, T299M, T299N, T299P,T299Q, T299R, T299S, T299V, T299W, T299Y, T335D, T335F, T335G, T335H,T335I, T335L, T335M, T335N, T335P, T335R, T335S, T335V, T335W, T335Y,V240A, V240I, V240I/V266I, V240M, V240T, V262A, V262E, V262F, V262I,V262T, V263A, V263I, V263M, V263T, V264D, V264E, V264E/N297D/I332E,V264F, V264G, V264H, V264I, V264I/A330L/I332E, V264I/A330Y/I332E,V264I/I332E, V264I/S298A/I332E, V264K, V264L, V264M, V264N, V264P,V264Q, V264R, V264S, V264T, V264W, V264Y, V266A, V266I, V266M, V266T,V266T, V273I, V273I, V282E, V282G, V282K, V282P, V282Y, V284E, V284L,V284N, V284T, V284Y, V302I, V302I, V303D, V303E, V303Y, V305E, V305T,V305Y, V323I, V323I, W313F, Y278D, Y278E, Y278G, Y278H, Y278I, Y278K,Y278L, Y278M, Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V, Y278W,Y296A, Y296D, Y296D/N297D/I332E, Y296E, Y296E/N297D/I332E, Y296G, Y296H,Y296H/N297D/I332E, Y296I, Y296K, Y296L, Y296M, Y296N, Y296N/N297D/I332E,Y296Q, Y296Q/N297D/I332E, Y296R, Y296S, Y296T, Y296T/N297D/I332E, Y296V,Y300A, Y300D, Y300E, Y300G, Y300H, Y300I, Y300K, Y300L, Y300M, Y300N,Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, and Y300W.
 34. A variantprotein according to claim 1 or 21 wherein said variant protein is anantibody comprising said Fc variant.
 35. A variant protein according toclaim 1 or 21 wherein said variant protein is an Fc fusion proteincomprising said Fc variant.
 36. A pharmaceutical composition comprisinga variant protein according to claim 1 and a pharmaceutically acceptablecarrier.
 37. A method of treating a mammal in need of said treatment,comprising administering a variant protein of claim 1.